Nucleic acid sequences for detecting genetic markers for cancer in a biological sample

ABSTRACT

Nucleic acid sequences for detecting the presence of nucleic acids, particularly mRNA, encoding human prostate-associated genetic markers encoding prostate-specific antigen (PSA), prostate specific membrane antigen (PSMA) or human kallikrein 2 (hK2) are disclosed. Preferred combinations of nucleic acid sequences amplifying and detecting the prostate-associated genetic markers RNA, used in methods that include amplification of the target sequences and detection of the amplified sequences are disclosed. Methods of detecting the presence of prostate-associated genetic marker nucleic acids, particularly mRNA, in a biological sample of non-prostate origin are disclosed.

This application is a divisional of application Ser. No. 11/838,168,filed Aug. 13, 2007, which is a divisional of U.S. Ser. No. 10/978,145,filed Oct. 28, 2004, now U.S. Pat. No. 7,267,956, which is a divisionalof U.S. Ser. No. 10/273,707, filed Oct. 17, 2002, now U.S. Pat. No.6,811,985, which is a divisional of U.S. Ser. No. 09/493,491, filed Jan.28, 2000, now U.S. Pat. No. 6,551,778, which claims the benefit under 35U.S.C. 119(e) of provisional application No. 60/117,640 filed on Jan.28, 1999, all of which applications are incorporated herein byreference.

FIELD OF THE INVENTION

This invention relates to detection of nucleic acids that serve ascancer markers in biological samples, and specifically relates tomethods for specifically detecting nucleic acids encodingprostate-specific antigen (PSA), prostate-specific membrane antigen(PSMA), and/or human glandular kallikrein (hK2) which, when presentindividually or together in a biological sample are indicative of thepresence of cancer in the organism, particularly prostate and breastcancers.

BACKGROUND OF THE INVENTION

Prostate cancer is a leading cause of death in men, particularly in menover 60 years of age. The biological aggressiveness of the cancer variesgreatly between individuals, with some cancers remaining as latenttumors which do not progress to clinical significance, and othersrapidly progressing and metastasizing to a fatal disease within a fewyears. Clinical diagnosis and staging of prostate tumors has relied ondigital rectal examination (DRE), computed tomography (CT) scans and/orendorectal magnetic resonance imaging (MRI). In addition, detection ofcells bearing molecular markers specific to or highly expressed inprostate tissue has been used diagnostically.

Prostate specific antigen (PSA) is a protease made in highconcentrations in prostatic epithelial cells and secreted into ducts ofthe prostate gland. PSA is a molecular marker for detection of prostatecancer (prostatic adenocarcinoma). Prostatic acid phosphatase (PAP) isanother secreted enzyme that has been used as a serum marker fordetection of prostatic metastases. A prostate-specific membrane antigen(PSMA), which is also highly expressed in prostate cancer tissuesincluding bone and lymph node metastases, has been characterized,isolated and the gene encoding it has been cloned (U.S. Pat. No.5,538,866 to Israeli et al.; O'Keefe D. S. et al, 1998, Biochim.Biophys, Acta 1443(1-2):113-127). Human glandular kallikrein-2 (hK2) isanother prostate-associated protein that has been linked to prostatecancer (Partin A. W. et al., 1999, Urology 54(5): 839-845; Darson M. F.et al., 1999, Urology 53(5): 939-944).

Researchers have also associated the presence of prostate-specificmarkers with breast tissue and/or breast secretions (Yu H. & Berkel H.,1999, J. La. State Med. Soc. 151(4): 209-213). For example, detection ofPSA in benign or malignant breast tumors and breast cyst fluids has beendemonstrated (Diamandis, E. P. et al., 1994, Breast Cancer Res. Treat32: 291-300; Yu, H. et al., 1994, Clin. Biochem. 27: 75-79; Monne, M. etal., 1994, Cancer Res. 54: 6344-6347; Malatesta M. et al., 1999, BreastCancer Res. Treat. 57(2): 157-163; Romppanen J. et al., 1999, Br. J.Cancer 79(9-10):1583-1587). Human kallikrein-2 is also expressed inbreast tumors and normal breast secretions (Black M. H. et al., 2000,Br. J. Cancer 82(2): 361-367). Breast cancer affects about 10% of theU.S. female population and, therefore, detection of cancer markersassociated with the disease has clinical utility.

Determining whether a cancer, such as prostate or breast cancer, isorgan-confined, locally invasive (i.e., for prostate cancer, penetratingthe capsule or seminal vesicle) or has metastasized to distant sites hassignificant impact on both the prognosis and determining the appropriatetreatment of the cancer. Therefore, effective methods of detectingcancer metastasis are medically important. For example, detectingmetastasis of prostate cancer to bone tissue or pelvic lymph nodes hasbeen used in staging the progress of the disease. Metastatic prostatecancer cells at these sites may be detected by histological examination,PSA-specific immunocytology, or by reverse transcriptase-polymerasechain reaction (RT-PCR) to detect PSA mRNA (Deguchi, T. et al., 1993,Cancer Res. 53: 5350-5354). Prostate cancer cells are also presumed tobe shed into the bloodstream, permitting them to disseminate to distantsites where the cells may become established as metastases. The presenceof detectable prostate-specific antigen (PSA) and/or PSA-synthesizingcells in circulating blood is an abnormal situation indicative ofpotential prostate cancer metastases (sometimes referred to as“hematogenous micrometastasis”), although only about 0.01% ofcirculating solid tumor cells eventually result in a metastatic deposit(Moreno, J. G. et al., 1992, Cancer Res. 52: 6110-6112). Similarly,detecting abnormal amounts of PSA in females may indicate the presenceof breast cancer (Yu H. & Berkel H., 1999, J. La. State Med. Soc.151(4): 209-213).

Monoclonal antibodies that react with various prostate tissue antigenshave been disclosed (U.S. Pat. No. 4,970,299 to Bazinet el al., U.S.Pat. No. 4,902,615 to Freeman et al., U.S. Pat. Nos. 4,446,122 and Re33,405 to Chu et al., U.S. Pat. No. 4,863,851 to McEwan et al., U.S.Pat. No. 5,055,404 to Ueda et al., U.S. Pat. No. 5,763,202 toHoroszewicz, and U.S. Pat. No. 5,773,292 to Bander). Monoclonalantibody-based immunoassays for measuring total PSA, free PSA (unboundto alpha-1-antichymotrypsin or “ACT”), and PSA-ACT complexes in bodyfluids have been disclosed for diagnostic methods to distinguish betweenpatients with benign prostatic hyperplasia (BHP) and those withprostatic carcinoma (U.S. Pat. No. 5,614,372 to Lilja et al.; U.S. Pat.Nos. 5,698,402 and 5,710,007 to Luderer et al.). Other knownimmunoassays measure total serum PSA and distinguish between free PSA inserum and PSA-protein complexes which tend to be in higherconcentrations in sera from prostate cancer patients (U.S. Pat. No.5,672,480 to Dowell et al.) PSA concentrations in amniotic fluid, asdetermined by antibody-based assays, have also been correlated withgestational times as an indicator of fetal abnormalities (U.S. Pat. No.5,579,534 to Diamandis).

RT-PCR detection of PSA-synthesizing cells in peripheral blood has alsobeen correlated with stage D1 to D3 pathology, and with capsularpenetration by prostate tumor cells (Moreno, J. G. et al., 1992, CancerRes. 52: 6110-6112; Katz, A. E. et al., 1994, Urology 43: 765-775; U.S.Pat. Nos. 5,506,106, 5,688,649 and 5,674,682 to Croce et al.; Vessella,R. L. et al., 1992, Proc. Am. Soc. Cancer Res. 33: Abstract No. 2367;Diamandis, E. P. & Yu, H., 1995, Clin. Chem. 41:177-179). Generally,RT-PCR assays rely on obtaining RNA from a blood sample, reversetranscribing the RNA into cDNA, amplifying the cDNA using a pair ofprimers complementary to separate regions of the PSA gene, anddemonstrating the presence of the amplified DNA by observing aparticular size DNA on a gel. PCR amplification of DNA requires arepeated series of thermal denaturation, primer annealing and synthesissteps (U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800159 to Mullis etal.). The amplified DNA may be further characterized by restrictionendonuclease digestion, probing with a PSA-specific oligonucleotide(e.g., Southern blotting), and/or DNA sequencing. Micrometastasis ofother types of solid tumors (melanoma, neuroblastoma, breast cancer,cervical cancer) has also been detected using RT-PCR assays for othercell markers (Wu, A. et al., 1990, US & Can. Acad. Pathol. Ann. Mtg.,Abstract No. 641; Smith, B. et al., 1991, Lancet 338: 1227-1229; Naito,H. et al., 1991, Eur. J. Cancer 27: 762-765; Mattano, Jr., L. A. et al.,1992, Cancer Res. 52: 4701-4705; U.S. Pat. Nos. 5,543,296 and 5,766,888to Sobel et al.).

PSA is a member of a group of serine proteases known as glandularkallikreins. The human kallikreins include pancreatic/renal kallikrein(hK1), prostate-specific glandular kallikrein (hK2 or HPSK), and PSA(also known as hK2) which are encoded by related genes (hKLK1, hKLK2 andhKLK3 or PSA, respectively). Because of the chemical and structuralsimilarities of these proteins and genes, it is important to be able todistinguish the individual proteins in immunoassays and the individualgenes or their corresponding mRNA in nucleic acid detection methods.Expression of hK2 has been associated with prostate and breast cancers(Black M. H. et al., 2000, Br. J. Cancer 82(2):361-367; Partin A. W. etal., 1999, Urology 54(5): 839-845; Darson M. F. et al., 1999, Urology53(5): 939-944). Antibodies that react specifically with humanprostate-specific glandular kallikrein (anti-hK2) but not with PSA havebeen described (U.S. Pat. No. 5,516,639 to Tindall et al.; U.S. Pat. No.5,786,148 to Bandman et al.). The gene sequences for human kallikreinsare known (U.S. Pat. No. 5,786,148 to Bandman et al.; GenBank AccessionNos. NM005551, M21895, M27274, M24543, M21897, M26663, M21896, S75755,U17040, S39329 and M8157).

Some nucleic acid sequences useful for amplification of PSA mRNA inRT-PCR assays and detection of DNA products have been described (e.g.,in Deguchi, T. et al., 1993, Cancer Res. 53: 5350-5354; Katz, A. E. etal., 1994, Urology 43: 765-775; Moreno, J. G. et al., 1992, Cancer Res.52: 6110-6112; U.S. Pat. Nos. 5,506,106, 5,688,649 and 5,674,682 toCroce et al.). Detection of prostate-associated genetic markers (e.g.,PSA, PSMA and/or hK2) at locations outside of prostate tissue is usefulfor detecting cancer metastases, particularly prostate cancer in men andbreast cancer in men and women, thereby indicating appropriatetreatment. Thus, there exists a clinical need for nucleic acid sequencesand methods that are used to specifically detect the presence of geneticexpression of prostate-associated genetic markers, i.e., specific mRNAsequences that provide diagnostic information. There is a particularneed for detecting these prostate-associated marker mRNA at levelsuseful for detecting relatively few cells containing the mRNA in abiological sample, such as occur in micrometastases.

SUMMARY OF THE INVENTION

The present invention is directed to assay methods for detecting and/orquantifying nucleic acids that encode prostate-specific antigen (PSA),particularly mRNA, in a non-prostate biological sample. In particular,the present invention includes preferred nucleic acid sequences usefulfor amplifying and hybridizing to amplified PSA-specific nucleic acids.

According to one aspect of the invention, there is provided anoligonucleotide having the sequence of any one of SEQ ID NO:1 to SEQ IDNO:43.

Another aspect of the invention is an oligonucleotide comprising atarget-binding sequence consisting of the sequence of any one of SEQ IDNO:15 to SEQ ID NO:43, and optionally a contiguous sequence required foran amplification reaction. In preferred embodiments, the contiguoussequence required for an amplification reaction is a sequence that is apolymerase binding sequence, and more preferably, the polymerase bindingsequence binds a T7 RNA polymerase. Preferred embodiments areoligonucleotides having the sequence is any one of SEQ ID NO:1 to SEQ IDNO:14.

Another aspect of the invention is a combination of oligonucleotidesused in a detection assay specific for a prostate specific antigen (PSA)target nucleic acid sequence. The combination comprises a firstoligonucleotide that serves as a first amplification primer thathybridizes specifically to a first PSA-specific sequence contained in anexon of a PSA expressed gene sequence, or that spans a joining pointlinking two exons of a PSA expressed gene sequence; a secondoligonucleotide that serves as a second amplification primer thatspecifically hybridizes to a different, non-overlapping secondPSA-specific sequence contained in an exon of a PSA expressed genesequence, or that spans a joining point linking two exons of a PSAexpressed gene sequence; and a third oligonucleotide that serves as adetection probe that specifically hybridizes to a third PSA-specificsequence contained in one or more exons of a PSA expressed genesequence. Preferably, the first PSA-specific sequence is contained inPSA exon 2, exon 3 or exon 4, or is a sequence that spans a joiningpoint linking exons 2 and 3 or exons 3 and 4. Preferably, the secondPSA-specific sequence is contained in PSA exon 3, exon 4 or exon 5, oris a sequence that spans a joining point linking exons 3 and 4 or exons4 and 5. Preferably, the third PSA-specific sequence is contained withinPSA exon 2, exon 3, exon 4 or exon 5, or spans a joining point thatlinks PSA exons 2 and 3, exons 3 and 4, or exons 4 and 5. Particularlypreferred combinations of oligonucleotides are a first oligonucleotidecomprising a sequence selected from the group consisting of SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21 and SEQ IDNO:26; the second oligonucleotide comprising a sequence selected fromthe group consisting of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:38, SEQ IDNO:40 and SEQ ID NO:41; and the third oligonucleotide comprises asequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, and SEQID NO:14. Additional preferred combinations of first, second and thirdoligonucleotides, in that order, have sequences of:

SEQ ID NO:15, SEQ ID NO:30 and SEQ ID NO:1; SEQ ID NO:17, SEQ ID NO:30and SEQ ID NO:1; SEQ ID NO:18, SEQ ID NO:30 and SEQ ID NO:1; SEQ IDNO:18, SEQ ID NO:35 and SEQ ID NO:1; SEQ ID NO:18, SEQ ID NO:30 and SEQID NO:4; SEQ ID NO:18, SEQ ID NO:30 and SEQ ID NO:5; SEQ ID NO:18, SEQID NO:30 and SEQ ID NO:6; SEQ ID NO:16, SEQ ID NO:31 and SEQ ID NO:2;SEQ ID NO:16, SEQ ID NO:31 and SEQ ID NO:3; SEQ ID NO:16 SEQ ID NO:33and SEQ ID NO:2; SEQ ID NO:16, SEQ ID NO:33 and SEQ ID NO:3; SEQ IDNO:16, SEQ ID NO:32 and SEQ ID NO:2; SEQ ID NO:16, SEQ ID NO:32 and SEQID NO:3; SEQ ID NO:16, SEQ ID NO:34 and SEQ ID NO:2; SEQ ID NO:16, SEQID NO:34 and SEQ ID NO:3; SEQ ID NO:20, SEQ ID NO:37 and SEQ ID NO:8;SEQ ID NO:20, SEQ ID NO:38 and SEQ ID NO:8; SEQ ID NO:21, SEQ ID NO:37and SEQ ID NO:8; SEQ ID NO:21, SEQ ID NO:38 and SEQ ID NO:8; SEQ IDNO:21, SEQ ID NO:40 and SEQ ID NO:14; SEQ ID NO:21, SEQ ID NO:37 and SEQID NO:14; SEQ ID NO:21, SEQ ID NO:41 and SEQ ID NO:14; SEQ ID NO:21, SEQID NO:40 and SEQ ID NO:8; SEQ ID NO:21, SEQ ID NO:41 and SEQ ID NO:8;SEQ ID NO:26, SEQ ID NO:37 and SEQ ID NO:8; SEQ ID NO:26, SEQ ID NO:40and SEQ ID NO:8; or SEQ ID NO:26 SEQ ID NO:41 and SEQ ID NO:8.

Particularly preferred combinations of first, second and thirdoligonucleotides, in that order, have sequences of: SEQ ID NO:17, SEQ IDNO:30 and SEQ ID NO:1;

SEQ ID NO:18, SEQ ID NO:30 and SEQ ID NO:1; SEQ ID NO:20, SEQ ID NO:37and SEQ ID NO:8; SEQ ID NO:21, SEQ ID NO:40 and SEQ ID NO:14; SEQ IDNO:21, SEQ ID NO:41 and SEQ ID NO:14; SEQ ID NO:26, SEQ ID NO:37 and SEQID NO:8; SEQ ID NO:26, SEQ ID NO:40 and SEQ ID NO:8; or

SEQ ID NO:26, SEQ ID NO:41 and SEQ ID NO:8. In other embodiments, thecombination of first, second and third oligonucleotides further includesat least one helper probe oligonucleotide. Preferred helper probeoligonucleotides included in a combination consist of an oligonucleotidehaving the sequence of SEQ ID NO:27 or SEQ ID NO:28, or a combination ofoligonucleotides having those sequences. Particularly preferredcombinations of first, second and third oligonucleotides and helperprobe oligonucleotides, in that order, have the sequences of:

SEQ ID NO:21, SEQ ID NO:41, SEQ ID NO:8 and SEQ ID NO:27; SEQ ID NO:21,SEQ ID NO:41, SEQ ID NO:8, SEQ ID NO:27 and SEQ ID NO:28; SEQ ID NO:26,SEQ ID NO:40, SEQ ID NO:8, SEQ ID NO:27 and SEQ ID NO:28; SEQ ID NO:26,SEQ ID NO:37, SEQ ID NO:8, SEQ ID NO:27 and SEQ ID NO:28; SEQ ID NO:26,SEQ ID NO:41, SEQ ID NO:8, SEQ ID NO:27 and SEQ ID NO:28; SEQ ID NO:26,SEQ ID NO:43, SEQ ID NO:8, SEQ ID NO:27 and SEQ ID NO:28; or

SEQ ID NO:26, SEQ ID NO:38, SEQ ID NO:8, SEQ ID NO:27 and SEQ ID NO:28.More preferably, the combinations of first, second and thirdoligonucleotides and helper probe oligonucleotides, in that order, havesequences of:

SEQ ID NO:21, SEQ ID NO:41, SEQ ID NO:8, SEQ ID NO:27 and SEQ ID NO:28;SEQ ID NO:26, SEQ ID NO:40, SEQ ID NO:8, SEQ ID NO:27 and SEQ ID NO:28,SEQ ID NO:26, SEQ ID NO:37, SEQ ID NO:8, SEQ ID NO:27 and SEQ ID NO:28;or SEQ ID NO:26, SEQ ID NO:41, SEQ ID NO:8, SEQ ID NO:27 and SEQ IDNO:28.

One aspect of the invention is a method of detecting aprostate-associated target nucleic acid in a biological samplecontaining nucleic acid, comprising the steps of providing a nucleicacid sample containing a target nucleic acid that includes at least aportion of at least one expressed gene sequence encodingprostate-specific antigen (PSA), prostate-specific membrane antigen(PSMA) or human kallikrein 2 (hK2); then hybridizing to the targetnucleic acid at least one primer oligonucleotide containing a sequencethat hybridizes specifically to the target nucleic acid or a complementthereof; producing a plurality of amplification products of the targetnucleic acid by using at least one polymerase activity; providing aprobe oligonucleotide that hybridizes specifically to at least oneamplification product of the target nucleic acid; and detecting a signalresulting from the probe hybridized to the amplification product. Inpreferred embodiments the at least one primer oligonucleotide comprisesthe sequence of any one of SEQ ID NO:15 to SEQ ID NO:29, SEQ ID NO:46,SEQ ID NO:48, or a target-binding sequence of any one of SEQ ID NO:30 toSEQ ID NO:43, SEQ ID NO:47 or SEQ ID NO:49. In one embodiment, thetarget nucleic acid is a PSA mRNA; the at least one primeroligonucleotide comprises a promoter-primer oligonucleotide consistingof a sequence complementary to at least a portion of the PSA mRNA and asequence that is a promoter sequence, and at least one primeroligonucleotide that hybridizes specifically to a nucleic acid strandcomplementary to the PSA mRNA; and the probe oligonucleotide hybridizesspecifically to amplification products of a sense that is the same asthat of the PSA mRNA. Preferably, the promoter-primer oligonucleotidecomprises the sequence of any one of SEQ ID NO:30 to SEQ ID NO:43, andthe primer oligonucleotide that hybridizes specifically to a nucleicacid strand complementary to the PSA mRNA comprises the sequence of anyone of SEQ ID NO:15 to SEQ ID NO:29. In another embodiment, the targetnucleic acid is a PSMA mRNA; the at least one primer oligonucleotidecomprises a promoter-primer oligonucleotide consisting of a sequencecomplementary to at least a portion of the PSMA mRNA and a sequence thatis a promoter sequence, and at least one primer oligonucleotide thathybridizes specifically to a nucleic acid strand complementary to thePSMA mRNA; and the probe oligonucleotide hybridizes specifically toamplification products of a sense that is the same as that of the PSMAmRNA. Preferably, the promoter-primer oligonucleotide comprises thesequence of SEQ ID NO:49, and the primer oligonucleotide that hybridizesspecifically to amplification products of a sense that is the same asthat of the PSMA mRNA comprises the sequence of SEQ ID NO:48. In anotherembodiment, the target nucleic acid is a hK2 mRNA; the at least oneprimer oligonucleotide comprises a promoter-primer oligonucleotideconsisting of a sequence complementary to at least a portion of the hK2mRNA and a sequence that is a promoter sequence, and at least one primeroligonucleotide that hybridizes specifically to a nucleic acid strandcomplementary to the hK2 mRNA; and the probe oligonucleotide hybridizesspecifically to amplification products of a sense that is the same asthat of the hK2 mRNA. Preferably, The method of claim 14, wherein thepromoter-primer oligonucleotide comprises the sequence of SEQ ID NO:47,and the primer oligonucleotide that hybridizes specifically toamplification products of a sense that is the same as that of the hK2mRNA comprises the sequence of SEQ ID NO:46. Preferably, the detectingstep uses at least one probe oligonucleotide consisting of a sequence ofany one of SEQ ID NO:1 to SEQ ID NO:14, SEQ ID NO:27, SEQ ID NO:28 orSEQ ID NO:50. One embodiment of the method comprises assaying for theexpressed gene sequence encoding PSA and at least one expressed genesequence encoding PSMA or hK2. In preferred embodiments, the nucleicacid sample is RNA, more preferably mRNA, isolated from human prostatetissue, peripheral blood, breast tissue, kidney tissue, small intestine,lung tissue, liver tissue or lymph node. In preferred embodiments, thedetecting step detects a signal in a homogeneous detection assay.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing showing the PSA gene structure andalignment of various PSA and kallikrein-1 sequences available fromGenBank and or EMBL using the accession numbers (Acc. No.) providedbelow, where the closed boxes (▪) indicate exons 1 to 5 (“E1”, “E2”,“E3”, “E4” and “E5”) and the open boxes (□) indicate introns (“I1”,“I2”, “I3” and “I4” are specifically labeled, whereas introns precedingthe first exon or following the last exon are not labeled), presentedover a scale representing the number of bases from 0 to 6,800 to showthe relative positions of the introns and exons. The diagramed known PSAgene sequences are labeled: “HSPSAR” (1,466 bp; Acc. No. X05332),“HUMAPS” (1,446 bp; Acc. No. M26663), “HSPSA” (1,729 bp; Acc. No.X07730), “HUMPAA” (1,415 bp; Acc. No. M21895), “HUMPAB” (1,654 bp; Acc.No. M21896), “HUMPAC” (658 bp; Acc. No. M21897), “S75755” (569 bp; Acc.No. S75755), “HSU17040” (990 bp; Acc. No. U17040), “HSPSA1” (389 bp;Acc. No. X13940), “HSPSA2” (287 bp; Acc. No. X13941), “HSPSA3” (372 bp;Acc. No. X13942), “HSPSA4” (281 bp; Acc. No. X13943), “HSPSA5” (900 bp;Acc. No. X13944), “HSPSAG” (5,873 bp; Acc. No. X14810), “HUMPSANTIG”(6,153 bp; Acc. No. M24543), and “HUMPSAA” (7,130 bp; Acc. No. M27274),and the human glandular kallikrein-1 sequence “S39329” (1,541 bp; Acc.No. S39329).

FIG. 2 is a schematic drawing showing the relative locations of variousprobes of the present invention (listed on the left using SEQ ID NO.)over a solid line representing the majority of the expressed PSA gene(based on the 1,466 bp sequence of HSPSAR, Acc. No. X05332) with thelocations of exons 1-5 (labeled E1, E2, E3, E4, and E5) shown above thesolid line and the splice junctions of deleted introns 1-4 (labeled I1,I2, I3, and I4) shown below the solid line.

FIG. 3 is a schematic drawing showing the relative locations of variousprobes of the present invention (listed on the left using SEQ ID NO.)over a solid line representing the majority of the expressed PSA gene(based on the 1,466 bp sequence of HSPSAR, Acc. No. X05332) with thelocations of exons 1-5 (labeled E1, E2, E3, E4, and E5) shown above thesolid line and the splice junctions of deleted introns 1-4 (labeled I1,I2, I3, and I4) shown below the solid line.

FIG. 4 is a schematic drawing showing the relative locations of variousprobes of the present invention (listed on the left using SEQ ID NO.)over a solid line labeled “HSPSAR” representing the majority of theexpressed PSA gene (based on the 1,466 bp sequence of HSPSAR, Acc, No.X05332) with the locations of exons 1-5 (labeled E1, E2, E3, E4, and E5)shown above the solid line and the splice junctions of deleted introns1-4 (labeled I1, I2, I3, and I4) shown below the solid line.

FIG. 5 is a diagram showing the linearity of PSA-specific targetamplification and detection of chemiluminescence (Net Mean RLU) inassays containing 10, 5, 1, 0.5, 0.1, 0.05, 0.01 or 0.005 pg of prostatetotal RNA per reaction, in which the experimental results are shown as adotted line and dotted symbols and the calculated linear regression lineis solid.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes nucleic acid sequences specific forsegments of a human PSA gene which are used in methods of detectingPSA-specific sequences in nucleic acids prepared from a biologicalsample that is not taken from prostate tissue. The invention furtherincludes nucleic acid sequences specific for segments of otherprostate-associated genetic markers, a human PSMA gene and hK2 gene,which are used in methods of detecting prostate-associated sequencesthat are useful cancer detection markers in nucleic acids prepared froma biological sample of non-prostate tissue, including breast tissue. Thenon-prostate tissue can include, for example, blood, lymph node, breastor breast cyst, kidney, liver, lung, muscle, stomach or intestinaltissue. The invention also includes preferred methods that combinenucleic acid sequences for amplifying and detecting PSA-specificsequences, PSMA and/or hK2 sequences, individually or in combination, innon-prostate tissue. The preferred methods amplify PSA, PSMA and/or hK2mRNA sequences without requiring thermal cycling and detect theamplified sequences in a homogeneous assay system. The methods mayoptionally include a simplified method of preparing RNA samples derivedfrom non-prostate tissue samples, such as peripheral blood, lymph nodesor bone marrow, although other non-prostate tissues may be used. Thepreferred RNA preparation method is relatively simple and provides RNAsuitable for analysis and detection of mRNA species that occur inrelatively low abundance in the biological samples.

The sequence of the human PSA gene is known and is available in a numberof databases (GenBank and EMBL databases, in Accession Nos. AF007544,X05332, M26663, X07730, M21895, M21896, M21897, S75755, U17040, X13942,X14810, M24543, M27274; accession numbers beginning with “X” indicatesequences in the EMBL database). The sequence of the related gene forthe human glandular kallikrein-1 is known (Acc. No. S39329) as is thehuman prostate-specific membrane antigen sequence (GenBank Acc. No.M99487). As shown in FIG. 1, the PSA and kallikrein-1 genes sharesimilar structural characteristics, which include five exons (labeled E1to E5) separated by four internal introns (labeled I1 to I4). The entirePSA gene sequence, including untranslated 5′ and 3′ regions, spans about6.8 kb. Because of the structural and sequence similarities of the PSAgene and the other members of the kallikrein gene family, theappropriate selection of PSA sequences to serve as PSA-specific primersand probes is critical to methods of amplifying and detectingPSA-specific nucleic acids in non-prostate tissue samples (i.e., toavoid false positives resulting from amplification and/or detection ofkallikrein nucleic acids). Preferred probes, primers andpromoter-primers of the present invention used for detectingPSA-specific sequences are shown in Table 1 (with their SEQ ID NOs).

TABLE 1 SEQ ID PSA NO: Nucleic Acid Sequence Length Location 1GGACCACCTGCTACGCCTCAG 21 Exon 3 2 GACCAAGTTCATGCTGTGTGCTG 23 Exon 4 3GACCAAGTTCATGCTGTGTGCTG 23 Exon 4 4 GCTGTGAAGGTCATGGACCTGCC 23 Exon 3 5GAACCAGAGGAGTTCTTGACCC 22 Exon 3/4 6 GGCCAGATGGTGCAGCCGGGAGC 23 Intron 37 GCAGTCTGCGGCGGTGTTCTG 21 Exon 2 8 ACAGCTGCCCACTGCATCAGG 21 Exon 2 9GTTCACCCTCAGAAGGTGACC 21 Exon 4 10 GCTGTGTGCTGGACGCTGGAC 21 Exon 4 11GCTTGTGGCCTCTCGTGGCAG 21 Exon 2 12 TGGCCTCTCGTGGCAGGGCAGT 22 Exon 2 13TCTCGTGGCAGGGCAGTCTGC 21 Exon 2 14 GTGCACCCCCAGTGGGTCCTC 21 Exon 2 15GATGCTGTGAAGGTCATGGACCTG 24 Exon 3 16 GTGCGCAAGTTCACCCTCAGAAGG 24 Exon 417 GAAGGTCATGGACCTGCCCACCCA 24 Exon 3 18 CTGTCAGAGCCTGCCGAGCTCACG 24Exon 3 19 GCTGCTCCGCCTGTCAGAGCCTG 23 Exon 3 20 GCTTGTGGCCTCTCGTGGCAG 21Exon 2 21 TCTCGTGGCAGGGCAGTCTGC 21 Exon 2 22 TTCCAATGACGTGTGTGCGCA 21Exon 4 23 GGAGGCTGGGAGTGCGAGAAGCAT 24 Exon 2 24 GGCTGGGAGTGCGAGAAGCATT22 Exon 2 25 TGGCCTCTCGTGGCAGGGCAGT 22 Exon 2 26 GCAGTCTGCGGCGGTGTTCTG21 Exon 2 27 GTGCACCCCCAGTGGGTCCTC 21 Exon 2 28 AACAAAAGCGTGATCTTGCTGGG23 Exon 2/3 29 CAAAAGCGTGATCTTGCTGGGT 22 Exon 3 30TAAATTAATACGACTCACTATAGGGAGACCAG 54 Exon 4 AGGGTGAACTTGCGCACACACG 31TAAATTAATACGACTCACTATAGGGAGACTGC 50 Exon 5 ACCACCTTGGTGTACAGG 32TAAATTAATACGACTCACTATAGGGAGACTCA 54 Exon 5 TGGTTCACTGCCCCATGACGTG 33AATTTAATACGACTCACTATAGGGAGATGCAC 48 Exon 5 CACCTTGGTGTACAGG 34AATTTAATACGACTCACTATAGGGAGACATGG 51 Exon 5 TTCACTGCCCCATGACGTG 35AATTTAATACGACTCACTATAGGGAGAGAGGG 50 Exon 3 TGAACTTGCGCACACACG 36TAAATTAATCGACTCACTATAGGGAGACCACC 52 Exon 4 TTCTGAGGGTGAACTTGCG 37TAAATTAATACGACTCACTATAGGGAGAGCCG 49 Exon 3 ACCCAGCAAGATCACGC 38TAAATTAATACGACTCACTATAGGGAGACTGT 49 Exon 3 GGCTGACCTGAAATACC 39TAAATTAATACGACTCACTATAGGGAGAGTGT 49 Exon 5 ACAGGGAAGGCCTTTCG 40TAAATTAATACGACTCACTATAGGGAGAACCC 50 Exon 3 AGCAAGATCACGCTTTTG 41TAAATTAATACGACTCACTATAGGGAGAAGGC 51 Exon 3 TGTGCCGACCCAGCAAGAT 42TAAATTAATACGACTCACTATAGGGAGACCTG 52 Exon 3 TGTCTTCAGGATGAAACAGG 43TAAATTAATACGACTCACTATAGGGAGACTGA 52 Exon 3 CCTGAAATACCTGGCCTGTG

FIGS. 2 to 4 show the relative locations of these sequences compared tothe expressed human PSA gene sequences (1,466 bp sequence of HSPSAR,Acc. No. X05332), diagramed with the relative locations of the exons andintron splice sites at the bottoms of the figures. Referring to FIG. 2,the relative locations of probes having sequences of SEQ ID NO:1 to SEQID NO:5 and SEQ ID NO:7 to SEQ ID NO:14 are shown. These preferredprobes are specific for PSA sequences in exons 2, 3 and 4. A probespecific for a PSA intron 3 sequence (SEQ ID NO:6) is not shown on FIG.2. Referring to FIG. 3, the relative locations of primers havingsequences of SEQ ID NO:15 to SEQ ID NO:29 are shown. These preferredprimers are specific for PSA sequences in exons 2, 3, 4, and spanningexons 2 and 3, including the splice junction (SEQ ID NO:28). Referringto FIG. 4, the relative locations of target-binding sequences ofpromoter-primers having sequences of SEQ ID NO:30 to SEQ ID NO:43 areshown. These preferred target-binding sequences are specific for PSAsequences in exons 3, 4 and 5.

For amplifying and detecting hK2 sequences, a preferred primer has thesequence GTCAGAGCCTGCCAAGATCACAG (SEQ ID NO:46) and a preferredpromoter-primer has the sequenceTAAATTAATACGACTCACTATAGGGAGACCACCAGCACACAACATGAACTCTGTC (SEQ ID NO:47).A labeled probe suitable for detecting the amplified hK2 sequences usesthe sequence of SEQ ID NO:1 shown in Table 1.

For amplifying and detecting PSMA sequences, a preferred primer has thesequence CAGATATGTCATTCTGGGAGGTC (SEQ ID NO:48) and a preferredpromoter-primer has the sequenceTAAATTAATACGACTCACTATAGGGAGACCAAATTCTTCTGCATCCCAGCTTGC (SEQ ID NO:49),and a labeled probe that includes the sequence CTCAGAGTGGAGCAGCTGTTGTTC(SEQ ID NO:50).

The present invention also includes a method for detecting andquantifying the PSA-specific RNA species, which is particularlyimportant because these mRNA species occur in relatively low abundancein RNA samples prepared from non-prostate tissues. Other embodiments ofthe invention include methods for detecting prostate-associated PSMA andhK2 RNA species, which when detected individually or in combination witheach other or PSA sequences, are important because these also are cancermarkers for prostate and breast cancer when found in non-prostatetissues. Moreover, detection of these prostate-associated markers (PSA,PSMA and hK2), individually and in combination, are clinically importantbecause cancers from individual patients may express one or more of themarkers, such that detecting one or more of the markers decreases thepotential of false negatives during diagnosis that might otherwiseresult if the presence of only one marker was tested. These methods areuseful for medical diagnoses without requiring a prostate biopsy and forclinically monitoring a patient's response to therapy for prostatecancer. Because the methods are sufficiently sensitive to detect thepresence of relatively low levels of prostate-associated RNA, such asPSA-specific mRNA, they are useful for detecting recurrence ormetastasis of prostate and/or breast cancer. Another advantage of thepresent methods is that amplification primers specific for differentexons of a target sequence will amplify only mRNA that have been splicedto link the exons in proximity, thereby eliminating amplification andfalse positive detection that might result from contaminating genomicDNA in the biological sample. In such an embodiment of the method,detection of genomic DNA is precluded because the exons in the genomicsequence are separated by intron sequence(s) such that the entire regionbetween the two primer binding sites will not be efficiently amplifiedand, therefore, will not be detected.

The invention also includes preferred combinations of nucleic acidsequences for amplifying and detecting human prostate-associated geneticmarkers, including those specific for PSA, PSMA and hK2 nucleic acidsequences.

In addition to definitions provided elsewhere in the specification, someterms have been defined as follows. Unless indicated or definedotherwise, all scientific and technical terms used herein have the samemeaning as commonly understood by those skilled in the relevant art.General definitions of many terms used herein are provided in Dictionaryof Microbiology and Molecular Biology, 2nd ed. (Singleton et al., 1994,John Wiley & Sons, New York, N.Y.). The Harper Collins Dictionary ofBiology (Hale & Marham, 1991, Harper Perennial, New York, N.Y.), andDorland's Illustrated Medical Dictionary, 27th ed. (W. A. Dorland, 1988,W.B. Saunders Co., Philadelphia, Pa.).

By “nucleotide sequence” or “nucleic acid sequence” is meant thesequence of nitrogenous bases along a linear information-containingmolecule that is capable of hydrogen-bonding with another nucleic acidstrand of DNA or RNA having a complementary base sequence. The terms arenot meant to limit such information-containing molecules to polymers ofnucleotides per se but also include molecule structures containing oneor more nucleotide analogs or abasic units in the polymer. The polymersmay include base subunits containing a sugar moiety or a substitute forribose or deoxyribose (for example, 2′ halide- or methoxy-substitutedpentose sugars), and may be linked by linkages other than phosphodiesterbonds, such as phosphorothioate, methylphosphonate, and peptidelinkages.

By “oligonucleotide” is meant a polymeric chain of two or more chemicalsubunits, each subunit comprising a nucleotide base moiety, a sugarmoiety, and a linking moiety which joins the subunits in a linearspacial configuration. An oligonucleotide may contain up to thousands ofsuch subunits, but generally contains subunits in a range having a lowerlimit of between about 5 to about 10 subunits, and an upper rangebetween about 20 to about 1,000 subunits. The most common nucleotidebase moieties are guanine (G), adenine (A), cytosine (C), thymine (T)and uracil (U), although other rare or modified nucleotide bases able toform hydrogen bonding (e.g., inosine or 1) are well known to thoseskilled in the art. The most common sugar moieties are ribose anddeoxyribose, although 2′-O-methyl ribose, halogenated sugars, and othermodified and different sugars are well known. The linking group isusually a phosphorus-containing moiety, commonly a phosphodiesterlinkage, although other known phosphate-containing linkages (e.g.,phosphorothioates, methylphosphonates) and non-phosphorus-containinglinkages (e.g. peptide-like linkages found in “peptide nucleic acids” orPNA) known in the art are included. Thus, PNA are intended to fallwithin this definition of an oligonucleotide. Likewise, anoligonucleotide includes one in which at least one base moiety has beenmodified, for example, by the addition of propyne groups, so long as (1)the modified base moiety retains the ability to form a non-covalentassociation with G, A, C, T or U, and (2) an oligonucleotide comprisingat least one modified nucleotide base moiety is not sterically preventedfrom hybridizing with a complementary single-stranded nucleic acid. Anoligonucleotide's ability to hybridize with a complementary nucleic acidstrand under particular conditions (e.g., temperature, saltconcentration) is governed by the sequence of base moieties, as is wellknown to those skilled in the art (Sambrook, J. et al., 1989, MolecularCloning, A Laboratory Manual, 2^(nd) ed. (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., particularly pp. 7.37-7.57 and11.47-11.57).

By “amplification” is meant production of multiple copies of a targetnucleic acid that contains at least a portion of a the intended specifictarget nucleic acid sequence (PSA, PSMA or hK2). The multiple copies maybe referred to as amplicons or amplification products. Preferably, theamplified target contains less than the complete target gene sequence(introns and exons) or an expressed target gene sequence (splicedtranscript of exons and flanking untranslated sequences). For example,PSA-specific amplicons may be produced by amplifying a portion of thePSA target polynucleotide by using amplification primers which hybridizeto, and initiate polymerization from, internal positions of the PSAtarget polynucleotide. Preferably, the amplified portion contains adetectable target sequence which may be detected using any of a varietyof well known methods.

By “nucleic acid amplification conditions” is meant environmentalconditions including salt concentration, temperature, the presence orabsence of temperature cycling, the presence of a nucleic acidpolymerase, nucleoside triphosphates, and cofactors which are sufficientto permit the production of multiple copies of a target nucleic acid orits complementary strand using a nucleic acid amplification method. Manywell-known methods of nucleic acid amplification require thermocyclingto alternately denature double-stranded nucleic acids and hybridizeprimers. For example, the polymerase chain reaction or PCR (U.S. Pat.Nos. 4,683,195; 4,683,202; 4,800,159; 4,965,188) uses multiple cycles ofdenaturation, annealing of a pair of primers to opposite strands, andprimer extension lead to exponential increases in copies of the targetsequence. In a variation called RT-PCR, reverse transcriptase (RT) isused to make a complementary DNA (cDNA) from mRNA, and the cDNA is thenamplified by PCR to produce multiple copies of DNA. The ligase chainreaction or LCR (Weiss, R. 1991, Science 254: 1292) uses two sets ofcomplementary DNA oligonucleotides which hybridize to adjacent regionsof the target nucleic acid and are covalently linked by using a DNAligase, in repeated cycles of thermal denaturation, hybridization andligation, to produce a detectable double-stranded ligatedoligonucleotide product. Another method is strand displacementamplification or SDA (Walker, G. et al., 1992, Proc. Natl. Acad. Sci.USA 89:392-396; U.S. Pat. Nos. 5,270,184 and 5,455,166) which usescycles of annealing pairs of primer sequences to opposite strands of atarget sequence, primer extension in the presence of a dNTP[α]S toproduce a duplex hemiphosphorothioated primer extension product,endonuclease-mediated nicking of a hemimodified restriction endonucleaserecognition site, and polymerase-mediated primer extension from the 3′end of the nick to displace an existing strand and produce a strand forthe next round of primer annealing, nicking and strand displacement,resulting in geometric amplification of product. Thermophilic SDA ortSDA uses thermophilic endonucleases and polymerases at highertemperatures in essentially the same method (European Pat. App. 0684,315). Other amplification methods include nucleic acid basedsequence amplification or NASBA (U.S. Pat. No. 5,130,238), one that usesan RNA replicase (Qβ replicase) to amplify the probe molecule itself(Lizardi, P. et al., 1988, BioTechnol. 6: 1197-1202), a transcriptionbased amplification method (Kwoh, D. et al., 1989, Proc. Natl. Acad.Sci. USA 86: 1173-1177), self-sustained sequence replication (Guatelli,J. et al., 1990, Proc. Natl. Acad. Sci. USA 87: 1874-1878), andtranscription mediated amplification (U.S. Pat. Nos. 5,480,784 and5,399,491 to Kacian & Fultz) which produces multiple RNA transcripts ofthe target sequence. For further discussion of known amplificationmethods, see Persing, David H., 1993, “In Vitro Nucleic AcidAmplification Techniques” in Diagnostic Medical Microbiology: Principlesand Applications (Persing et al., Eds.), pp. 51-87 (American Society forMicrobiology, Washington, D.C.).

Preferred transcription based amplification systems of the presentinvention employ an RNA polymerase to make many RNA transcripts of atarget region (U.S. Pat. Nos. 5,480,784 and 5,399,491 to Kacian &Fultz). Transcription mediated amplification (TMA) uses apromoter-primer that hybridizes to a target nucleic acid in the presenceof a reverse transcriptase and an RNA polymerase to form a doublestranded promoter. Then, RNA polymerase activity produces RNAtranscripts that can become templates for further rounds of TMA in thepresence of a second primer capable of hybridizing to the RNAtranscripts. Unlike PCR, LCR or other methods that require heatdenaturation, TMA is an isothermal method that uses an RNAse H activityto digest the RNA strand of an RNA-DNA hybrid, thereby making the DNAstrand available for hybridization with a primer or promoter-primer.Generally, the RNAse H activity associated with a retroviral reversetranscriptase provided for amplification is used.

By “primer” or “amplification primer” is meant an oligonucleotidecapable of binding to a region of a target nucleic acid or itscomplement and promoting nucleic acid amplification of the targetnucleic acid. In most cases a primer will have a free 3′ end which canbe extended by a nucleic acid polymerase. All amplification primersinclude a base sequence capable of hybridizing via complementary baseinteractions either directly with at least one strand of the targetnucleic acid or with a strand that is complementary to the targetsequence. Amplification primers serve as substrates for enzymaticactivity that produces a longer nucleic acid product.

For example, in PCR, amplification primers anneal to opposite strands ofa double stranded target DNA that has been denatured and the primers areextended by a thermostable DNA polymerase to produce double-stranded DNAproducts which are denatured with heat, cooled and annealed to theamplification primers, and the primers are extended by polymeraseactivity during multiple cycles (e.g., about 20 to about 50 thermiccycles).

In another example, in TMA, one amplification primer is a“promoter-primer” that hybridizes to a target RNA and reversetranscriptase (RT) produces a cDNA copy of the target RNA, while RNase Hactivity degrades the target RNA. The promoter-primer is anoligonucleotide that comprises a promoter sequence (which becomefunctional when double stranded) located to 5′ of a target-bindingsequence which is capable of hybridizing to the target sequence. Thetarget-binding sequence is capable of hybridizing to a binding site ofthe target RNA at a location 3′ to the sequence to be amplified. Whenmade double-stranded, the promoter sequence is capable of binding an RNApolymerase to begin transcription of the target sequence to which thepromoter primer is hybridized. A promoter-primer may be referred to as a“T7-primer” when it is specific for T7 RNA polymerase recognition. Undercertain circumstances the 3′ end of a promoter-primer, or asubpopulation of such promoter-primers, may be modified to block orreduce primer extension. A second amplification primer then binds to thecDNA and RT produces another DNA strand resulting in a double-strandedDNA containing a functional RNA promoter at one end. The secondamplification primer comprises a target-binding sequence capable ofhybridizing to the complement (i.e., the cDNA strand) of the target RNA.It may be referred to as a “non-T7 primer” to distinguish it from a“T7-primer”. An RNA polymerase uses this promoter sequence to producemultiple RNA transcripts (i.e., amplicons), generally about 100 to 1,000copies. Each newly-synthesized amplicon can anneal with the secondamplification primer which is extended by RT to produce a DNA copy,while the RNase H activity degrades the RNA of this RNA:DNA duplex. Thepromoter-primer then binds to the newly synthesized DNA and RT creates adouble-stranded DNA from which the RNA polymerase produces multipleamplicons. A billion-fold isothermic amplification can thus be achievedusing two amplification primers.

A “target-binding sequence” of an amplification primer is the portionthat determines target specificity because that portion is capable ofannealing to the a target nucleic acid strand or its complementarystrand. The complementary target sequence to which the target-bindingsequence hybridizes is referred to as a primer-binding sequence. Forprimers or amplification methods that do not require additionalfunctional sequences in the primer (e.g., PCR amplification), the primersequence consists essentially of a target-binding sequence, whereasother methods (e.g., TMA or SDA) include additional specializedsequences adjacent to the target-binding sequence (e.g., an RNApolymerase promoter sequence adjacent to a target-binding sequence in apromoter-primer or a restriction endonuclease recognition sequence foran SDA primer). For example, in the preferred PSA-specifictarget-binding sequences shown in Table 1, SEQ IN NO:15 to SEQ ID NO:29are amplification primers that do not include additional functionalsequences, and SEQ ID NO:30 to SEQ ID NO:43 are T7 promoter-primers thatinclude additional functional sequences (underlined) besides thetarget-binding sequences (not underlined); the underlined sequences arepreferred T7 polymerase promoter sequences. It will be appreciated bythose skilled in the art that all of the primer and probe sequences ofthe present invention may be synthesized using standard in vitrosynthetic methods. Also, it will be appreciated that those skilled inthe art could modify primer sequences disclosed herein using routinemethods to add additional specialized sequences (e.g., promoter orrestriction endonuclease recognition sequences) to make primers suitablefor use in a variety of amplification methods. Similarly,promoter-primer sequences described herein can be modified by removingthe promoter sequences to produce amplification primers that areessentially target-binding sequences, suitable for amplificationprocedures that do not use these additional functional sequences, suchas PCR.

The PSA-specific sequences of the primers and probes of SEQ ID NO:1 toSEQ ID NO:43 and the hK2-specific sequences of primers of SEQ ID NO:46AND 47 were selected by aligning the known sequences of PSA exons withthe known sequences of HSPSAR and human kallikrien exon sequences(HUMKAL2, HUMKAL2a, HUMKAL2b, HUMKAL2c, HUMKAL2d, HSKALLI, and HUMPSAA)using the an alignment algorithm using default parameters of thealgorithm, such as, for example, the BLASTN algorithm available from theNational Center for Biotechnology Information at the National Library ofMedicine, or using the Multiple Alignment Construction and AnalysisWorkbench (“MACAW”) algorithm as described in detail previously (Shuleret al., 1991, Proteins 9(3):180-190). After these sequences werealigned, regions of the PSA exon or hK2 region for which a primer orprobe sequence was desired which contained the least amount of identitywith the other sequences were identified. From those sequences,oligonucleotide sequences of the appropriate length and GC content for aprimer or probe were selected and synthesized for tested. In some cases,the predicted secondary structure of the selected primer or probesequence was determined by using an algorithm that calculates RNAsecondary structure, as previously described in detail (Matzura andWennborg, Complete Applications in the Biosciences, 1996, Vol. 12, No.3, pp 247-9). The primers of SEQ ID NO:48 and SEQ ID NO:49 and the probeof SEQ ID NO:50 for the human PSMA were similarly designed and selected.Although algorithms were used to aid in sequence alignment andpredicting secondary structure, those skilled in the art could readilyperform these steps manually.

By “target sequence” is meant the nucleotide base sequence of a nucleicacid strand, at least a portion of which is capable of being detectedusing a labeled oligonucleotide probe. Primers bind to a portion of atarget sequence, which includes both complementary strands when thetarget sequence is a double-stranded nucleic acid.

By “equivalent RNA” is meant a ribonucleic acid having the samenucleotide base sequence as a deoxyribonucleic acid (DNA), with theappropriate U for T substitution(s), Similarly, an “equivalent DNA” is aDNA having the same nucleotide base sequence as an RNA but with theappropriate T for U substitution(s). It will be appreciated by thoseskilled in the art that the terms “nucleic acid” and “oligonucleotide”refer to molecular structures having either a DNA or RNA base sequenceor a synthetic combination of DNA and RNA base sequences, includinganalogs thereof, which include “abasic” residues.

By “solid support” is meant a material that is essentially insolubleunder the solvent and temperature conditions of the assay method,comprising free chemical groups available for joining an oligonucleotideor nucleic acid. Preferably, the solid support is covalently coupled toan oligonucleotide designed to directly or indirectly bind a targetnucleic acid. When the target nucleic acid is an mRNA, theoligonucleotide attached to the solid support is preferably a poly-Tsequence. A preferred solid support is a particle, such as a micron- orsubmicron-sized bead or sphere. A variety of solid support materials arecontemplated, such as, for example, silica, polyacrylate,polyacrylamide, a metal, polystyrene, latex, nitrocellulose,polypropylene, nylon or combinations thereof. More preferably, the solidsupport is capable of being attracted to a location by means of amagnetic field, such as a solid support having a magnetite core.Particularly preferred supports are monodisperse magnetic spheres (i.e.,uniform size±about 5%).

By “detecting” an amplification product is meant any of a variety ofmethods for determining the presence of an amplified nucleic acid, suchas, for example, hybridizing a labeled probe to a portion of theamplified product. A labeled probe is an oligonucleotide thatspecifically binds to another sequence and contains a detectable groupwhich may be, for example, a fluorescent moiety, a chemiluminescentmoiety, a radioisotope, biotin, avidin, enzyme, enzyme substrate, orother reactive group. Preferably a labeled probe includes an acridiniumester (AE) moiety that can be detected chemiluminescently underappropriate conditions (as described in U.S. Pat. No. 5,283,174). Otherwell know detection techniques include, for example, gel filtration, gelelectrophoresis and visualization of the amplicons, and High PerformanceLiquid Chromatography (HPLC). The detecting step may either bequalitative or quantitative, although quantitative detection ofPSA-specific or other prostate-associated genetic amplicons is preferredfor determining the level of prostate-associated gene expression (e.g.,PSA-specific mRNA) in a non-prostate sample, which indicates the degreeof metastasis or recurrence of prostate and/or breast cancer.

Assays for purifying and detecting a target polynucleotide often involvecapturing a target polynucleotide onto a solid support. The solidsupport retains the target polynucleotide during one or more washingsteps of the target polynucleotide purification procedure. Onehybridization sandwich technique for capturing and for detecting thepresence of a target polynucleotide involves the capture of the targetpolynucleotide by a probe bound to a solid support and hybridization ofa detection probe to the captured target polynucleotide (U.S. Pat. No.4,486,539 to Ranki et al.). Detection probes not hybridized to thetarget polynucleotide are readily washed away from the solid support.Thus, remaining label is associated with the target polynucleotideinitially present in the sample. Another method uses a mediatorpolynucleotide that hybridizes to both a target polynucleotide and to apolynucleotide fixed on a solid support such that the mediatorpolynucleotide joins the target polynucleotide to the solid support toproduce a bound target (U.S. Pat. No. 4,751,177 to Stabinsky). A labeledprobe can be hybridized to the bound target and unbound labeled probecan be washed away from the solid support.

Many methods for detecting mRNA, particularly those that includeamplification, require extensive purification of RNA and/or mRNA priorto amplification and detection, often involving harsh chemicals such asguanidinium thiocyanate (Sambrook, J. et al., 1989, Molecular Cloning, ALaboratory Manual, 2^(nd) ed. (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.), pp. 7.37-7.57; Lin, L. et al., 1993, “Simple andRapid Sample Preparation Methods for Whole Blood and Blood Plasma” inDiagnostic Molecular Microbiology, Principles and Applications (Persing,D. H. et al., Eds., American Society for Microbiology, Washington,D.C.), pp 605-616). One embodiment of the present invention uses asimple and rapid method for non-prostate biological sample preparationthat results in RNA from which prostate-associated mRNA (e.g.,PSA-specific mRNA) can be amplified and detected for detection ofprostate and/or breast cancer cells in non-prostate tissue. Non-prostatebiological samples may be hematopoietic tissue such as peripheral bloodor bone marrow, plasma, non-prostate biopsy tissue including lymphnodes, respiratory tissue or exudates, gastrointestinal tissue, urine,feces, semen or other body fluids or materials. The preferred samplepreparation method requires a minimum of technical expertise and usesstandard laboratory equipment and relatively low cost reagents to yieldtarget mRNA suitable for amplification, while avoiding false positivesthat may result from potentially cross-reactive sequences found inchromosomal DNA. The preferred simplified sample preparation methodeliminates extensive extraction and shearing of chromosomal DNA (toreduce viscosity), avoids use of potentially harmful reagents (e.g.,guanidinium compounds, phenol or chloroform) and minimizes the number ofsteps, thus minimizing sample loss and increasing detection of lowabundance mRNA species.

The methods invention includes a method of detecting or quantifyingprostate-associated genetic markers, such as PSA-specific nucleic acids,particularly mRNA. The methods include contacting a non-prostatebiological sample that potentially contains a prostate-associatedgenetic marker target sequence with a first primer or promoter-primercapable of specifically hybridizing to the target gene sequence (PSA,PSMA and/or hK2), and providing at least one nucleic acid polymeraseactivity, under nucleic acid amplification conditions to producetarget-specific amplification products (“amplicons”). Preferably, thetarget-specific amplification products are a plurality of a nucleic acidstrands comprising a region complementary to the specific targetsequence and at least one probe binding site capable of hybridizingunder hybridization conditions with a labeled oligonucleotide probespecific for the amplified target sequences, thereby forming aprobe:target hybrid. The method also includes detecting the labeledprobe:target hybrid as an indication of the presence of theprostate-associated genetic marker nucleic acid in the non-prostatebiological sample. Preferred embodiments further include preparation ofthe target nucleic acid substrate from the biological sample using arelatively simple sample preparation procedure. The detecting step mayfurther include one or more helper probes specific for a portion of theamplified target nucleic acid to enhance detection of the target in theassay. Although not wishing to be bound by the mechanism by which thehelper probe(s) enhance detection, it is generally thought that helperprobes hybridize to the amplified target sequences to remove secondarystructure or otherwise enhance the capability of the other probes tobind to the detected sequence. Preferably, if the prostate-associatedgenetic marker target sequence is RNA, then the labeled oligonucleotideprobe is of the same sense.

In one embodiment of the present method, only a promoter-primer is usedin the amplifying step and the probe is targeted to a portion of theamplicon. When a single promoter-primer is employed with no primer ofthe opposite sense, the target RNA is detected using a probe of the samesense as the target RNA and capable of hybridizing with a base sequenceregion located within the amplified complementary nucleic acid (aspreviously described in detail in U.S. Pat. No. 5,554,516 to Kacian etal.).

In preferred embodiments of the present invention, selected primersspecific for human PSA, PSMA and/or hK2 gene sequences are used in atranscription mediated amplification that uses both a promoter-primerand an amplification primer without promoter sequence, and the amplifiedproducts are detected with a probe capable of being detected in ahomogeneous assay (see U.S. Pat. Nos. 5,480,784 and 5,399,491 to Kacian& Fultz; U.S. Pat. No. 5,554,516 to Kacian et al.; U.S. Pat. No.5,283,174 to Arnold et al.; European Pat. App. No. EP 0709466). Thesegeneral amplification and detection methods are well known in the art.The preferred amplification method produces an amplified nucleic acid oramplicon that is an RNA, and even more preferably produces predominantlyamplified RNA that is complementary to the target sequence (i.e., theopposite sense as the target). Thus, if the target is mRNA, arbitrarilydesignated (+) sense strands, then the amplicons are preferably (−)sense strands. The probes for detecting such amplicons are any sequencecapable of hybridizing specifically to an amplified sequence (i.e., arecomplementary to a portion of the amplicon). Usingtranscription-mediated amplification (“TMA”) methods as describedpreviously (U.S. Pat. Nos. 5,480,784 and 5,399,491), the promoter-primeris of the (−) sense the template target nucleic acid is of the (+)sense, and the produced amplicons are of the (−) sense.

This method is substantially isothermal and is preferable to prior artmethods for detecting PSA-specific sequences because it does not requiretemperature cycling for nucleic acid amplification or nested primers forPSA-specific serial amplification. In particularly preferred embodimentsof the present method, one of the two primers used in the amplificationstep is a T7 (− sense) promoter-primer that binds to theprostate-associated genetic marker mRNA and one of the two primers is anon-T7 (+ sense) primer that binds to the cDNA product produced from theactivity of reverse transcriptase extending the T7 promoter-primer thatuses the mRNA as a template. The T7 promoter-primer contains a T7 RNApolymerase recognition sequence at the 5′ end of the target-bindingsequence, whereas the non-T7 primer does not contain a T7 RNA polymeraserecognition sequence and consists essentially of a target-bindingsequence. Preferred promoter-primers have a length range having a lowerlimit of between about 35-45 nt and an upper limit between about 45-100nt. Preferably, promoter-primers are about 40-70 nt long, and morepreferably are about 45-55 nt long. Preferably, a T7 promoter sequenceof a promoter-primer is about 25-30 nt long; particularly preferred T7promoter sequences include SEQ ID NO:44 or SEQ ID NO:45. Preferredtarget-binding regions of a PSA-specific promoter-primer hybridizespecifically to a human PSA gene exon sequence, preferably within anyone of exons 3, 4 or 5. Primers used in transcription mediatedamplification in conjunction with promoter primers consist essentiallyof target-binding sequences specific for human PSA, PSMA or hK2 genesequences. Preferred PSA-specific bind to PSA sequences within an exonor spanning a splice junction of two exons, and more preferably havetarget-binding sequences specific for any one of exons 2, 3 or 4, orspan the exon 2 and 3 splice junction of the human PSA gene. Primerspreferably have a length within a range having a lower limit of about15-20 nt and an upper limit within a range of about 25-100 nt, morepreferably in a range of about 15-30 nt, and most preferably in a rangeof about 20-25 nt in length. As shown in Table 1, preferred PSA-specificprimers have the sequences of SEQ ID NO:15 to SEQ ID NO:29 and preferredPSA-specific promoter-primers have the sequences of SEQ ID NO:30 to SEQID NO:43, where the promoter sequence is underlined and thetarget-binding sequence is not underlined.

One aspect of the present invention is a method of amplifying aPSA-specific target sequence in mRNA present in a sample taken fromnon-prostate tissue and detecting the amplified product as an measure ofprostate or breast cancer cells present in non-prostate tissue, thusindicating the presence of breast cancer or metastasis of prostatecancer. Although a homogeneous chemiluminescent detection assay fordetecting the nucleic acid probe is a preferred embodiment, thoseskilled in the art could readily use other labels and detection methodswell known in the art, such as labels based on enzymes, enzymesubstrates, fluorescent, luminescent, chemiluminescent andelectrochemiluminescent molecules, radionuclides, and fluorescent atomsto detect a hybridized probe; the amplification product may be detectedusing standard methods such as by gel electrophoresis or filtration,increased light absorption, hyperchromatic shift, or HPLC. Preferredhomogeneous assays have advantages over heterogeneous assays (i.e.,those necessitating physical separation to differentiate signal ofhybridized probe from signal due to unhybridized probe), but thehomogeneous detection aspect is not critical to the present invention.Preferably a fluorescent or chemiluminescent label is incorporated intothe probe, and more preferably the label is a chemiluminescentacridinium ester (AE).

Probes for use in the methods of the present invention may be targetedto any region of the amplicon to be detected, and preferably the probeis of the same sense as the target nucleic acid. For example, aPSA-specific probe may hybridize to either on intron or exon sequence,and preferably hybridizes to a sequence within a PSA gene exon (e.g., inexon 2, 3 or 4), or spanning an exon splice site (e.g., spanning the 3′end of exon 3 and the 5′ end of exon 4), or within a PSA gene intron(e.g., within intron 3). Probe sequences may be within a range of about5-100 nt long, and preferably are within about 10-50 nt long, morepreferably about 20-25 nucleotides long. Preferred probe sequencesinclude those having SEQ ID NO:1 to SEQ ID NO:14 (with their generallocation in the human PSA gene shown in Table 1) and SEQ ID NO:50.Preferably, the probes are AE-labeled and hybridize to the amplified RNAproducts of a TMA reaction.

The primers and probes of the present invention may be used inamplification and detection methods that use nucleic acid substratesisolated by any of a variety of well known methods. The target mRNA mayprepared by the following relatively simple procedure to yield mRNAsuitable for use in TMA. In this procedure, cells in a biological sample(e.g., peripheral blood or bone marrow cells) were lysed by contactingthe cell suspension with a lysing solution containing at least about 150mM of a soluble salt, preferably a lithium halide salt, a chelatingagent and a non-ionic detergent in an effective amount to lyse thecellular cytoplasmic membrane without causing substantial release ofnuclear DNA or RNA. The cell suspension and lysing solution were mixedat a ratio of about 1:1 to 1:3, and the detergent concentration in thelysing solution was between about 0.5% to 1.5% (v/v). Any of a varietyof known non-ionic detergents are effective in the lysing solution(e.g., TRITON®-type, TWEEN®-type and NP-type); typically the lysingsolution contained an octylphenoxy polyethoxyethanol detergent,preferably 1% TRITON® X-102. This procedure has been used primarily withbiological samples that contain cell suspensions (e.g., blood and bonemarrow), but it works equally well on other tissues if the cells areseparated using standard mincing, screening and/or proteolysis methodsto separate cells individually or into small clumps. After cell lysis,the released total RNA was stable, and may be stored at room temperaturefor at least 2 hr without significant RNA degradation without additionalRNAse inhibitors. Total RNA may be used in amplification without furtherpurification, or mRNA may be isolated using standard methods, generallydependent on affinity binding to the poly-A portion of mRNA.

Preferably mRNA isolation used capture particles consisting essentiallyof poly-dT oligonucleotides attached to insoluble particles that wereadded to the above-described lysis mixture, the poly-dT moietiesannealed to the poly-A mRNA, and the particles were separated physicallyfrom the mixture. Generally, superparamagnetic particles were used andseparated by applying a magnetic field to the outside of the container.Preferably, a suspension of about 300 μg of particles (in a standardphosphate buffered saline (PBS), pH 7.4, of 140 mM NaCl) having eitherdT₁₄ or dT₃₀ linked at a density of about 1 to 100 pmoles per mg(preferably 10-100 pmols/mg, more preferably 10-50 pmols/mg) were addedto about 1 ml of lysis mixture. Any superparamagnetic particles may beused, although typically the particles were a magnetite core coated withlatex or silica (e.g., commercially available from Serodyn or Dynal) towhich poly-dT oligonucleotides were attached using standard procedures(Lund et al., Nuc. Acids Res., 1988, 16:10861-10880). The lysis mixturecontaining the particles was gently mixed and incubated at about 22° C.to 42° C. for about 30 min, when a magnetic field was applied to theoutside of the tube to separate the particles with attached mRNA fromthe mixture and the supernatant was removed. The particles were washedone or more times, generally three, using standard resuspension methodsand magnetic separation as described above. Then, the particles weresuspended in a buffer solution and used immediately in amplification orstored frozen.

A number of parameters may be varied without substantially affecting thesample preparation. For example, the number of particle washing stepsmay be varied and the particles may be separated from the supernatant byother means (e.g., filtration, precipitation, centrifugation). The solidsupport may have nucleic acid capture probes joined thereto that arecomplementary to the specific target sequence or any particle or solidsupport that non-specifically binds the target nucleic acid may be used(e.g., polycationic supports as described in U.S. Pat. No. 5,599,667 toArnold et al.). For amplification, the isolated RNA was released fromthe capture particles using a standard low salt elution process oramplified while retained on the particles by using primers that bind toregions of the RNA not involved in base pairing with the poly-dT or inother interactions with the solid phase matrix. The exact volumes andproportions described above are not critical and may be varied so longas significant release of nuclear material does not occur. Vortex mixingis preferred for small scale preparations but other mixing proceduresmay be substituted. But it is important that samples derived frombiological tissue be treated to prevent coagulation, and the ionicstrength of the lysing solution be at least about 150 mM, preferably 150mM to 1M, because lower ionic strengths lead to nuclear materialcontamination (e.g., DNA) that increases viscosity and may interferewith amplification and/or detection steps to produce false positives.Lithium salts are preferred in the lysing solution to prevent RNAdegradation, although other soluble salts (e.g., NaCl) combined with oneor more known RNAse inhibitors would be equally effective.

Table 2 lists preferred combinations of primers and probes used foramplification and detection of PSA, PSMA and hK2 genetic markers, withor without helper probe(s), using TMA essentially as describedpreviously in U.S. Pat. Nos. 5,399,491, 5,480,784, and 5,554,516, anddetection in a homogeneous protection assay, essentially as describedpreviously in U.S. Pat. No. 5,283,174. Each row represents a preferredcombination. Of these, particularly preferred combinations foramplifying and detecting PSA mRNA are indicated with an asterisk (*) ineach box of the row in Table 2. The last two entries in Table 2 areprimers and probes for amplifying and detecting specifically hK2 target(SEQ ID NOS 46, 47 and 1) and PSMA target (SEQ ID NOS 48, 49 and 50).

Nucleic acids from a variety of biological samples have been used intesting the methods of prostate-associated marker mRNA amplification anddetection with the primers and probes described herein, including invitro transcripts of a PSA cDNA, whole blood spiked with in vitrotranscripts of a PSA cDNA, total RNA isolated from a prostate cancercell line (LNCaP cells, Horoszewicz J. S. et al., 1983, Cancer Res. 43:1809-1818; ATCC No. CRL-10995), whole blood spiked with LNCaP cells,peripheral blood, breast tissue cells, lung cells, poly-A RNA isolatedand tested individually from prostate cancer cells, lymph nodes, breasttissue cells, kidney cells, small intestine tissue cells and white bloodcell genomic DNA. Total RNA (from lung, mammary gland and prostate) andpoly-A RNA (from kidney, liver, lymph node, mammary gland, smallintestine and prostate) were prepared using standard methods (derivedfrom the method of Chirgwin et al., 1979, Biochem. 18:5294, with poly-ARNA selected using oligo(dT) cellulose; commercially available fromClontech Laboratories, Inc., Palo Alto, Calif.). Total RNA and poly-ARNA were prepared from peripheral blood from normal human donors usingsubstantially the same methods.

In vitro transcripts of a PSA cDNA were prepared using a PSA cDNA clone(obtained from the ATCC, Accession No. 106527) present in a vector(pBluscript SK−; Adams et al., 1985, Nature 377(3): 174) or the PSA cDNAfragment of about 1.2 kb was subcloned into another vector (pSP64poly(A) vector) for use in preparing in vitro transcripts. The plasmidDNA was prepared and purified using standard methods, linearized byenzymatic digestion at a point 3′ of the cDNA insert, and transcribedusing an RNA polymerase. Transcripts were prepared using standardmethods and reagents (supplied in an AMPLISCRIBE Translation Kit, fromEpicenter Technologies, Corp., Madison, Wis.).

TABLE 2 Preferred Combinations of Primers and Probes (SEQ ID NOs) Non-T7primer T7 promoter-primer Probe Helper Primer(s) 15 30 1 NONE  17*  30* 1* NONE  18*  30*  1* NONE 18 35 1 NONE 18 30 4 NONE 18 30 5 NONE 18 306 NONE 16 31 2 NONE 16 31 3 NONE 16 33 2 NONE 16 33 3 NONE  16*  32*  2*NONE 16 32 3 NONE 16 34 2 NONE 16 34 3 NONE  20*  37*  8* NONE 20 38 8NONE 21 37 8 NONE 21 38 8 NONE  21*  40* 14* NONE 21 37 14  NONE  21* 41* 14* NONE 21 40 8 NONE 21 41 8 NONE 21 41 8 27  21*  41*  8* 27 &28*  26*  40*  8* 27 & 28*  26*  37*  8* 27 & 28*  26*  41*  8* 27 & 28*26 43 8 27 & 28  26 38 8 27 & 28  46 47 1 NONE 48 49 50  NONE

Unless mentioned otherwise, the techniques employed or contemplatedherein are standard methodologies well known to one of ordinary skill inthe art. The examples of embodiments that follow are provided forillustration only.

EXAMPLE 1 Lysis of Biological Samples and Isolation of mRNA

About 250 μl of uncoagulated peripheral blood or bone marrow was addedto about 750 μl of the lysing solution. The proportions of each of thesetwo components is not critical, and generally a 1:1 ratio to 1:3 ratioof the components is capable of lysing the samples. The lysing solutionused most commonly consisted of 50 mM HEPES (pH 7.5), 1 M LiCl, 5 mMEDTA, and 1% TRITON® X-102. By “uncoagulated” is meant that the blood orbone marrow was treated upon collection with about 2 mM to about 20 mMEDTA, or an effective amount of heparin or similar anticoagulant knownin the art. To this mixture, a suspension of about 300 μg ofsuperparamagnetic particles (in PBS solution, pH 7.4, containing 140 mMNaCl) having either dT₁₄ or dT₃₀ linked at a density of about 10 to 50pmoles of poly dT per mg of particles was added. The lysis mixturecontaining the poly-dT particles was gently mixed by vortexing andincubated at between about 22° C. to 42° C. for about 30 min. Theparticles with attached mRNA were separated from the mixture by applyinga magnetic field to the outside of the tube and removing thesupernatant. The particles were washed one to three times byresuspending them in about 1 ml of a wash solution (50 mM HEPES, pH 7.5,5 mM EDTA, 150 mM NaCl and 0.1% (w/v) sodium dodecyl sulfate (SDS)) withmixing by vortexing for about 3 to 5 seconds to suspend the beads, whichwere separated from the supernatant as described above, and thesupernatant wash was discarded. Following washing, the particles weresuspended in 250 μl of a buffer (10 mM HEPES, pH 7.5, 1 mM EDTA) andeither stored at −30° C. for later use or used immediately. For use inamplification procedures, the particles with attached RNA were useddirectly or, occasionally, the attached RNA was released from thecapture particles using a standard elution procedures (e.g., with heat).The presence of solid particles did not impair amplification and thesimplicity of using the particles with attached RNA was preferred to anadditional RNA isolation step.

EXAMPLE 2 Amplification and Detection of PSA-Specific mRNA

To initially test the relative efficiency of different combinations ofprimers and promoter-primers in transcription mediated amplification ofknown PSA sequences, individual combinations of a non-T7(+) primer and aT7(−) promoter-primer were used to amplify an in vitro transcript (asdescribed above) of PSA gene sequences. Following amplification, theamplification products were detected in a homogeneous protection assayessentially as described in U.S. Pat. No. 5,283,174, to Arnold et al.,using a single probe labeled with acridinium ester, in which thechemiluminescent signal is detected as relative light units (“RLU”). Allexperimental samples were tested in triplicate, and the mean RLU of thethree tests was calculated. For these experiments, the promoter-primerand the probe were the same in all reactions, but the non-T7 primerswere varied. The T7 promoter-primer (SEQ ID NO:30) is specific for PSAexon 4, the primers are specific for PSA exon 3 (SEQ ID NO:15, SEQ IDNO:17 and SEQ ID NO:18), and the probe is specific for PSA exon 3 (SEQID NO:1).

Briefly, 50 μl of a solution containing different numbers of in vitrotranscripts (10³, 10⁴, or 10⁵ molecules, or a negative controlcontaining no transcripts) was added to a tube containing 25 μl of anamplification reagent containing 160 mM Tris-HCl (pH 7.5), 100 mM MgCl₂70 mM KCl, 20% (w/v) polyvinylpyrrolidone, 16 mM each of the fourribonucleoside triphosphates ATP, GTP, CTP, and UTP, 4 mM each of thefour deoxyribonucleoside triphosphates dATP, dGTP, dCTP, and dTTP, 400nM (15 pmoles). In the three combinations tested in this example, the T7promoter-primer at 15 pmols/reaction (equivalent to about 400 nM) hadthe sequence of SEQ ID NO:30, and the non-T7 primer at 400 nM (15pmoles) had the sequence of SEQ ID NO:15, SEQ ID NO:17, or SEQ ID NO:18.Each reaction was incubated at 60° C. for 10 min, although anytemperature capable of melting intramolecular base pairing in the targetnucleic acid is suitable (e.g., between about 60° C. and 70° C., morepreferably between about 65° C. and about 67° C.). Then, each reactionwas incubated at about 42° C. for 5 min, after which an enzyme reagent(25 μl containing 2000 units of recombinant MMLV reverse transcriptase,2000 units recombinant T7 RNA polymerase, 8 mM HEPES (pH 7.5), 50 mMN-acetyl-L-cysteine, 0.04 mM zinc acetate, 80 mM trehalose, 140 mMTris-HCl (pH 8.0), 70 mM KCl, 1 mM EDTA, 0.01% (w/v) phenol red, 10%(v/v) TRITON® X-102 and 20% (v/v) glycerol) was added and the reactionwas gently mixed and incubated for about 1 hr at 42° C. Thisamplification method produced amplified RNA of a sense opposite to thatof the target RNA.

Amplified RNA was detected using 100 μl of a probe reagent containing100 mM lithium succinate (pH 4.7), 1.2 M LiCl, 15 mM aldrithiol-2, 2%(w/v) lithium lauryl sulfate (LLS), 20 mM EDTA, 20 mM ethyleneglycol-bis-(β-amino ethyl ether) N,N,N′,N′-tetracetic acid (EGTA), 3%ethanol, and 7.5 nM of a hybridization probe having SEQ ID NO:1, labeledwith a chemiluminescent acridinium ester (AE) linked via non-nucleotidelinkers using methods essentially a described in U.S. Pat. No. 5,585,481to Arnold et al. The detection solution was added to the reaction andincubated at 60° C. for 30 min to permit hybridization of the probe tothe amplified target.

The probe (SEQ ID NO:1) was detected in a homogeneous protection assay(HPA) which included the following steps. To hydrolyze the AE label onunbound probe, 300 μl of an alkaline solution (600 mM sodium borate, pH8.5, and 1% (v/v) TRITON® X-100) was added to the mixture describedabove. The AE label on hybridized probe is protected from hydrolysis byits association with a double helix, whereas the AE label onunhybridized probe is not protected from hydrolysis, making unhybridizedprobe undetectable. The solution was incubated at 60° C. for 10 min,cooled at room temperature for 5 min, and mixed with 200 μl of asolution containing 30 mM hydrogen peroxide and 1 mM nitric acid,followed immediately by addition of 200 μl of a solution containing 1 MNaOH and 2% (w/v) ZWITTERGENT® 3-14. Chemiluminescence was detectedusing a luminometer (e.g., LEADER® 50), with the output measured inrelative light units (“RLU”).

The results of these tests are shown in Table 3, in which the meannumber of RLU detected for each of three tests for each combination ofprimer and promoter-primer is presented. Each combination of primer andpromoter-primer was tested using no PSA transcripts (negative control)and individually with 10³, 10⁴, or 10⁵ transcripts.

TABLE 3 PSA Target Amplification and Detection with DifferentCombinations of Primers Non-T7 Primer T7 Promoter-primer Mean SEQ ID NO(PSA SEQ ID NO (PSA PSA Transcripts Signal exon) exon)(molecules/reaction) (RLU) 15 (exon 3) 30 (exon 4) 10⁵ 504,097 15 30 10⁴56,481 15 30 10³ 5,528 15 30  0 950 17 (exon 3) 30 (exon 4) 10⁵ 31,65617 30 10⁴ 3,409 17 30 10³ 1,217 17 30  0 983 18 (exon 3) 30 (exon 4) 10⁵1,490,310 18 30 10⁴ 1,451,391 18 30 10³ 505,344 18 30  0 901

The results shown in Table 3 show that the three combinations of primersand promoter-primers tested in this example gave varying levels ofamplification, all detected with the same probe, and all provided asignal above background when 10³ copies of the target RNA were includedin the assay. Of these three combinations, the combination of SEQ IDNO:18 and SEQ ID NO:30 gave the best results, and the system becamesaturated for detection when 10⁴ copies of the target RNA were includedin the assay.

EXAMPLE 3 Amplification and Detection of PSA mRNA in Total and Poly-ARNA from Prostate Tissue

In this example, a number of combinations of primer, promoter-primer andprobe were used to amplify and detect PSA-specific mRNA contained withina sample of total RNA prepared from prostate tissue as described above.

The transcription mediated amplification and chemiluminescence detectionsteps were performed substantially as described in Example 2. Initially,to determine the sensitivity of the assay, total RNA was used over arange of 1 pg to 10 ng, representing the RNA content of differentnumbers of prostate cells. That is, the total RNA in the tested sampleswas equivalent to a calculated numbers of cells as follows: 1 pg isequivalent to less than 1 cell (about 0.1 cell), 10 pg is equivalent toabout 1 cell, 100 pg is equivalent to about 10 cells, 1,000 pg (1 ng) isequivalent to about 100 cells, and 10,000 pg (10 ng) is equivalent toabout 100 cells. In some experiments, the total range of RNAconcentrations were not tested. A negative control (background) of noadded RNA was included in all tests, and all tests were performed intriplicate with the mean RLU reported in Table 4, Table 4 shows theresults for a number of combinations of non-T7 (+) primers and T7(−)promoter-primers used in amplification, and detected with a number ofAE-labeled probes.

TABLE 4 PSA Target Amplification and Detection with DifferentCombinations of Primers and Probes T7 promoter- Non-T7 Primer primerProbe SEQ ID NO SEQ ID NO SEQ ID NO Total RNA (PSA Exon) (PSA Exon) (PSAExon) Added Mean RLU 18 (exon 3) 30 (exon 4)  1 (exon 3) 10 ng 1,592,09218 30  1 1 ng 1,395,934 18 30  1 100 pg 966,851 18 30  1 10 pg 352,47618 30  1 1 pg 50,249 18 30  1 0 520 20 (exon 2) 37 (exon 3)  8 (exon 2)1 ng 2,490,049 20 37  8 0 4,780 21 (exon 2) 40 (exon 3) 14 (exon 2) 10pg 1,061,753 21 40 14 1 pg 130,779 21 40 14 0.1 pg 41,248 21 40 14 032,866 21 (exon 2) 37 (exon 3) 14 (exon 2) 10 pg 108,811 21 37 14 1 pg52,603 21 37 14 0.1 pg 34,963 21 37 14 0 37,440 21 (exon 2) 41 (exon 3)14 (exon 2) 10 pg 3,188,114 21 41 14 1 pg 237,928 21 41 14 0.1 pg 60,46021 41 14 0 37,140 21 (exon 2) 40 (exon 3)  8 (exon 2) 10 pg 107,787 2140  8 1 pg 36,444 21 40  8 0.1 pg 8,735 21 40  8 0 5,953 21 (exon 2) 37(exon 3)  8 (exon 2) 10 pg 67,169 21 37  8 1 pg 13,555 21 37  8 0.1 pg7,833 21 37  8 0 5,037 21 (exon 2) 41 (exon 3)  8 (exon 2) 10 pg 261,24921 41  8 1 pg 44,013 21 41  8 0.1 pg 6,308 21 41  8 0 5,242

As can be seen from the results shown in Table 45 all of thecombinations of primers, promoter-primers and probes detected PSA mRNAin the total RNA sample added to the reaction compared to the backgroundsignal for each combination, where no RNA was added. Furthermore, asignal significantly greater than background was generally detected whenas little as 1 pg of RNA was added (equivalent to about the amountcalculated to be present in 0.1 cell).

These results were confirmed in separate tests using poly-A RNA isolatedfrom prostate tissue. The prostate poly-A RNA was isolated using poly-dThybridization in which the poly-dT oligonucleotides were about dT₁₄ todT₃₀ in length and covalently linked to magnetic particles; thepurification steps used (magnetic separation and washing) weresubstantially as described in Example 1. It was expected that totalprostate RNA would contain about 5% PSA-specific mRNA, and the amount ofpoly-A RNA (i.e., purified mRNA) added to the amplification anddetection samples was reduced accordingly. In one set of tests, poly-ARNA from prostate tissue was added to triplicate tubes at 5 pg or 0.5 pgper reaction, which were amplified and detected using the combination ofSEQ ID NO:18 and SEQ ID NO:30 as primers, and SEQ ID NO:1 as probe,substantially as described for the results presented in Table 4. For 5pg of prostate mRNA, the mean RLU detected were 1,404,286 and for 0.5 pgthe mean RLU detected were 392,558. These results show that furtherpurification of the PSA-specific target, although not necessary foramplification and detection, produces a detectable signal with as littleas 0.5 pg of mRNA.

To determine whether helper primers added to an amplification reactionwould increase the signal detected, a series of amplification anddetection experiments were performed as described in the next example.

EXAMPLE 4 Amplification and Detection of PSA-Specific Target with HelperProbes

This example demonstrates that addition of one or more helper probes mayincrease the signal detected, allowing detection of PSA-specificsequences present in less than 0.1 pg of total RNA isolated fromprostate tissue using the preferred amplification and detection system.In these tests, amplification was performed substantially as describedin Examples 2 and 3, except that about 100 pmol of helper probeoligonucleotides were added to the amplification reaction along with theAE-labeled probe using in the detection step. Amplification thenproceeded as described above, followed by detection of chemiluminescenceas described above. The PSA-specific target was provided in total RNAisolated from prostate tissue as described in Example 3, tested atconcentrations ranging from 0.016 pg to 10 pg. The helper probes inthese experiments were SEQ ID NO:27 (in PSA exon 2) and SEQ ID NO:28(spanning the joining point of PSA exons 2 and 3). The results of thesetests are shown in Table 5, with the signal representing the mean oftriplicate samples for each RNA concentration tested.

TABLE 5 Amplification with Helper Probes and Detection of PSA-specificSequences Non-T7 T7 promoter- Primer primer Probe RNA Added SEQ ID NOSEQ ID NO SEQ ID NO Per Reaction (PSA Exon) (PSA Exon) (PSA Exon) (pg)Mean RLU 21 (exon 2) 41 (exon 3) 8 (exon 2) 10 7,539,550 21 41 8 11,065,253 21 41 8 0.5 422,459 21 41 8 0.25 174,277 21 41 8 0.125 126,54321 41 8 0.062 39,283 21 41 8 0.031 24,898 21 41 8 0.016 5,504 21 41 80.000 3,645

To show that the amplification and detection system was specific for PSAmRNA detection, amplification and detection reactions were performed ontotal RNA isolated from prostate tissue (obtained from Clonetech, PaloAlto, Calif.) compared to total RNA isolated from white blood cells(“WBC”) obtained from peripheral blood of normal donors. Helper probes(SEQ ID NO:27 and SEQ ID NO:28) were included in all of theseamplification reactions at a concentration of about 100 μmol and fourdifferent combinations of non-T7 primer, T7 promoter-primer and probe.In these tests, the prostate total RNA was used at 10 pg or 1 pg perreaction, whereas at least 10,000-fold more WBC total RNA was used forcomparison (1 μg or 100 ng per reaction). For each set of reactions, anegative control with no added RNA was included, and all reactions weretested in triplicate, with the results presented as the mean of thethree reactions (Mean RLU). The results are presented in Table 6.

TABLE 6 Specificity of Amplification with Helper Probes and Detection ofPSA-specific Sequences T7 promoter- Total RNA Non-T7 Primer primer ProbeAdded SEQ ID NO SEQ ID NO SEQ ID NO Per Reaction & Mean (PSA Exon) (PSAExon) (PSA Exon) Source RLU 21 (exon 2) 41 (exon 3) 8 (exon 2) 10 pg -Prostate 5,217,830 21 41 8 1 pg - Prostate 441,752 21 41 8 1 μg - WBC4,667 21 41 8 100 ng - WBC 16,136 21 41 8 0 2,552 26 (exon 2) 40 (exon3) 8 (exon 2) 10 pg - Prostate 9,085,005 26 40 8 1 pg - Prostate2,708,971 26 40 8 1 μg - WBC 7,417 26 40 8 100 ng - WBC 6,124 26 40 8 02,641 26 (exon 2) 37 (exon 3) 8 (exon 2) 10 pg - Prostate 3,709,715 2637 8 1 pg - Prostate 462,262 26 37 8 1 μg - WBC 18,889 26 37 8 100 ng -WBC 5,122 26 37 8 0 2,592 26 (exon 2) 41 (exon 3) 8 (exon 2) 10 pg -Prostate 2,710,422 26 41 8 1 pg - Prostate 439,147 26 41 8 1 μg - WBC8,409 26 41 8 100 ng - WBC 4,738 26 41 8 0 2,676

As can be seen from the results shown in Table 6, each of the fourcombinations of primers and probes tests specifically amplified anddetected PSA-specific target RNA in prostate total RNA. In contrast, theresults were essentially negative for the samples under the sameconditions that included WBC total RNA from a normal donor which wouldnot be expected to contain PSA-specific target mRNA. That is, despitethe use of 10,000-fold to 1,000,000-fold more WBC total RNA in thereactions compared to prostate total RNA, no false positives wereobserved (i.e., none gave results equivalent to those obtained with 1 pgof prostate RNA). The relatively high mean RLU (16,136 Mean RLU)presented in Table 6 for the tests using 100 ng of WBC total RNA and thecombination of SEQ ID NO:21, SEQ ID NO:41 and SEQ ID NO:8 resulted froma single tube in which 43,485 RLU were detected, compared to the othertwo tubes in the set (2,458 and 2,464 RLU) which were essentially thesame as the negative control; the single high result may have been dueto contamination or operator error.

In separate tests, the linearity of detection of PSA-specific targetsequence was measured in an amplification and detection assay in whichthe combination of non-T7(+) primer of SEQ ID NO:26, T7 promoter primerof SEQ ID NO:40 and probe of SEQ ID NO:8 was used with prostate totalRNA over a range of 10 pg to 0.001 pg per reaction (i.e., using 10, 5,1, 0.5, 0.1, 0.05, 0.01 or 0.005 pg per reaction). The test aliquotswere prepared from the 10 pg aliquot by diluting 1:2 and 1:10 to obtaina 5 pg and 1 pg aliquots; the 5 pg aliquot was then serially diluted1:10 to obtain the 0.5, 0.05 and 0.005 pg aliquots and the 1 pg aliquotwas similarly diluted to obtain the 0.1 and 0.01 pg aliquots. All assayswere performed in triplicate, and the negative control contained noprostate total RNA in the reaction. The results of these assays areshown graphically in FIG. 5, in which both the X-axis and the Y-axis arelogarithmic scales; by “net mean RLU” is meant that the mean of thenegative control results was subtracted from the mean RLU of theexperimental samples. Referring to FIG. 5, the experimental results areshown with dotted symbols and a dotted line, and the calculated linearregression of these results in shown as a solid line, with a fit of R²equal to 0.9776. When results within a series of 10-fold serialdilutions of total RNA were similarly graphed and R² values werecalculated, within the 10, 1, 0.1 and 0.01 pg group the R² value was0.9997 and within the 5, 0.5, 0.05 and 0.005 pg group the R² value was0.9879. These results show that the amplification and detection systemdescribed herein is quantitative for detection of PSA-specific targetnucleic acids.

EXAMPLE 5 Amplification and Detection of PSA Target Sequences inDenatured Genomic DNA

To show that the negative results obtained in Example 4 with RNAisolated from peripheral WBC were due to the lack of PSA gene expressionin WBC, amplification and chemiluminescent detection assays wereperformed using denatured and undenatured genomic DNA obtained fromperipheral WBC. The genomic DNA is expected to contain normal PSA genesequences, which would be inaccessible to primers for amplification orprobe for detection in its undenatured state. In these experiments thenon-T7 (+) primer was SEQ ID NO:18, the T7 promoter-primer was SEQ IDNO:30 and the AE-labeled probe was SEQ ID NO:30. The positive controlwas PSA gene in vitro transcripts prepared substantially as in Example 2and used as 100 copies per reaction, 10 copies per reaction and 1 copyper reaction. The negative control was a reaction run without additionof nucleic acid target (DNA or RNA). The WBC genomic DNA was isolatedusing standard DNA isolation procedures, with shearing to decreaseviscosity in solution (Sambrook et al., Molecular Cloning, A LaboratoryManual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1989), and used with or without prior denaturation atabout 5 μg, 0.5 μg and 0.05 μg per reaction. The DNA was denatured byheating to 95° C. for about 10 min and then cooled immediately.Amplification and detection assays were performed substantially asdescribed in Examples 2 and 3, and results of triplicate tests for eachsample are presented as mean RLU in Table 7.

TABLE 7 Amplification and Detection of PSA Target Sequence in WBCDenatured Genomic DNA Nucleic Acid Sample Nucleic Acid Per Reaction MeanRLU WBC Undenatured DNA 5 μg 18,234 WBC Undenatured DNA 0.5 μg 21,429WBC Denatured DNA 5 μg 1,391,494 WBC Denatured DNA 0.5 μg 293,522 PSA InVitro Transcripts 100 copies 1,457,098 PSA In Vitro Transcripts 10copies 448,603 PSA In Vitro Transcripts 1 copy 1,028 None None 1,068

The results shown in Table 7 show that the WBC genomic DNA contained PSAgene sequences which were amplified and detected when the DNA wasdenatured, with a decrease in signal consistent to the decrease inamount of target added to the reaction. In contrast, WBC undenatured DNAgave a relatively high background that was essentially the same at bothconcentrations tested, but was at least 10-fold less than the signaldetected with 0.5 μg WBC denatured DNA under the same conditions.

EXAMPLE 6 Amplification of Prostate Poly-A RNA and PSA In VitroTranscripts

This example compares amplification and detection results using apreferred combination of primers and probe, for which the target nucleicacid was either poly-A RNA isolated from prostate tissue or in vitrotranscripts of a PSA cDNA sequence. The prostate poly-A RNA was isolatedusing hybridization to poly-dT oligomers immobilized to a solid support,substantially as described in Examples 1 and 3, and the in vitrotranscripts were prepared substantially as described in Example 2. Theamplification and detection methods were performed substantially asdescribed in Examples 2-4, using as a non-T7(+) primer anoligonucleotide having SEQ ID NO:16, as a T7(−) promoter-primer anoligonucleotide having SEQ ID NO:32 and as a probe an oligonucleotidehaving SEQ ID NO:2, labeled with AE. The target sequences were added tothe amplification reactions, which were performed in triplicate for eachcombination, at a calculated concentration of 10,000 copies of in vitrotranscript per tube or 0.875 ng of poly-A-containing prostate RNA pertube, or no added RNA in the negative control tubes. The mean resultswere: 4,022 RLU for the negative control, 8,858 RLU for the in vitrotranscript samples, and 1,791,197 for the poly-A RNA samples. That is,although the detected chemiluminescence in these tests was relativelylow compared to other examples, the combination of SEQ ID NO:16, SEQ IDNO:32 and SEQ ID NO:2 was capable of amplifying and detectingPSA-specific target nucleic acid in the two positive samples (PSA invitro transcripts and prostate mRNA) compared to the negative control.

EXAMPLE 7 Amplification and Detection of PSA-Specific Target in ClinicalPeripheral Blood Samples

In this example, clinical samples of peripheral blood were tested fromindividuals suspected of having either benign prostate hyperplasia orprostate cancer. Peripheral blood samples were collected shortly beforeprostate surgery (pre-surgery) and soon after surgical removal of theprostate gland (post-surgery). For comparison, known numbers of cells ofa prostate cancer cell line (LNCaP cells; ATCC No. CRLI-10995) orPSA-specific in vitro transcripts (as described in Example 2) were mixedwith peripheral blood obtained from a normal individual. The clinical orcontrol blood samples were lysed and the mRNA was isolated using thesystem of poly-dT oligomers attached to magnetic particles substantiallyas described in Example 1. About 1.2 ml of lysate of clinical sampleswas used per assay; and about 1.0 ml of lysate for LNCaP cells was usedfor comparison. The amplification and detection methods were performedsubstantially as described in Examples 2-4, using as a non-T7(+) primeran oligonucleotide having SEQ ID NO:18, as a T7(−) promoter-primer anoligonucleotide having SEQ ID NO:30 and as a probe an oligonucleotidehaving SEQ ID NO:1, labeled with AE. The target sequences were added tothe amplification reactions, which were performed in triplicate for eachsample, at: (1) about 50 to 100 ng of total RNA, equivalent to about 2.5to 5 ng of mRNA per reaction for each clinical sample, (2) a calculatedconcentration of 2,000 or 200 or 20 copies of in vitro transcript perreaction, and (3) the calculated equivalent of about 10 cells, 1 cell or0.1 LNCaP cell per reaction. Negative control reactions contained noadded RNA. The mean RLU results for the triplicate samples for each setof reactions, or mean RLU of nine reactions for the negative controls,are presented in Table 8.

TABLE 8 Amplification and Detection of PSA-specific Target mRNA inPeripheral Blood Samples RNA Target Amount Mean or Equivalent RLU Sampleper Reaction Detected Blood + PSA In Vitro Transcripts 2,000 copies1,243,606 Blood + PSA In Vitro Transcripts   200 copies 190,850 Blood +PSA In Vitro Transcripts   20 copies 25,877 Clinical Sample 1,pre-surgery 2.5-5.0 ng poly A RNA 53,053 Clinical Sample 1, post-surgery2.5-5.0 ng poly A RNA 58,858 Clinical Sample 2, pre-surgery 2.5-5.0 ngpoly A RNA 1,436 Clinical Sample 2, post-surgery 2.5-5.0 ng poly A RNA1,346 Clinical Sample 3, pre-surgery 2.5-5.0 ng poly A RNA 2,354Clinical Sample 3, post-surgery 2.5-5.0 ng poly A RNA 1,447 ClinicalSample 4, pre-surgery 2.5-5.0 ng poly A RNA 1,286 Clinical Sample 4,post-surgery 2.5-5.0 ng poly A RNA 1,329 Blood + LNCaP cells ≈10 LNCaPcells 1,087,426 Blood + LNCaP cells ≈1.0 LNCaP cell 200,122 Blood +LNCaP cells ≈0.1 LNCaP cell 21,807 Normal Blood (Negative Control) None1,330

Based on these results, it is clear that the primers and probe arecapable of detecting PSA-specific target RNA less than the equivalent ofone cancer cell in a volume of about 1.0 ml peripheral blood as detectedin the sample containing the equivalent of one LNCaP cell. Based on theblood samples containing in vitro PSA gene transcripts, it is clear thatthe assay is capable of detecting as little as 20 copies of transcriptin a blood sample. By comparison with the results obtained in thesepositive controls and the negative controls to which no target RNA wereadded, the RLU detected in Clinical Sample No. 1 shows the presence ofPSA-specific target in the peripheral blood, indicative of prostatecancer in the patient which is shedding cells into the blood and mayhave metastasized. In contrast, Clinical Sample Nos. 2 to 4 did notcontain detectable PSA-specific nucleic acid in this assay, indicativeof benign prostate hyperplasia (“BPH”) instead of prostate cancer inthose patients. For all of the clinical samples tested, the levels ofRLU detected in pre-surgery and post-surgery samples obtained fromindividual patients were not significantly different. In those patientswith RLU levels indicative of BPH, this would be an expected. In thepatient who provided Clinical Sample No. 1, the presence of detectablePSA-specific target RNA in the peripheral blood shortly after surgeryindicates that cells expressing PSA mRNA, presumably prostate cancercells, are still present in peripheral blood, indicative of shed cancercells or metastasis.

In further testing of clinical samples using the same assay system todetect PSA-specific target RNA in peripheral blood, a total of 30samples were tested: 5 from normal controls, 15 from patients clinicallydiagnosed as having prostate cancer and 10 clinically diagnosed withBPH. Each reaction contained about 500 ng of total RNA, and each samplewas assayed in triplicate. Samples in which greater than 5,000 mean RLUwere detected were scored as positive for prostate cancer, and otherswere scored as negative for prostate cancer. All of the normal controlsamples and 9 of the 10 samples from BPH patients were negative. Samplesthat were positive for PSA-specific target RNA included 11 of the 15samples from patients clinically diagnosed as having prostate cancer andone of the BPH-diagnosed patients. These results show that the assay forPSA-specific target in peripheral blood was consistent with the clinicalsymptoms in the majority of cases. For those patients diagnosed withprostate cancer where PSA-specific target was not detected in blood, thecancer may not have metastasized or shed cells into the blood.

EXAMPLE 8 Amplification and Detection of PSA-Specific Target in VariousBiological Samples

Although prostate specific antigen (PSA) has been associated with cancerin prostate tissue, amplification and detection assays were performed onRNA isolated from other human tissues to determine the relative levelsof PSA-specific target detected in those tissues. Unless otherwiseindicated, all tissues were obtained from normal donors (i.e.,non-cancerous tissue). The assays were performed in triplicate for eachsample using either total RNA or poly-A RNA isolated from human tissuesamples substantially as described in previous examples using anon-T7(+) primer an oligonucleotide having SEQ ID NO:18, a T7(−)promoter-primer an oligonucleotide having SEQ ID NO:30 and an AE-labeledprobe have the oligonucleotide sequence of SEQ ID NO:1. The results ofthese assays are presented in Table 9, with the negative control resultsbeing the mean of 12 reactions performed without adding target RNA.

TABLE 9 Detection of PSA-specific Target in Biological Samples AmountTested Biological Sample Per Reaction Mean RLU Prostate Tissue Total RNA100 ng 1,695,965 Prostate Tissue Total RNA 10 ng 1,832,916 ProstateTissue Total RNA 1 ng 1,170,802 Peripheral Blood Total RNA 100 ng 2,341Breast Tissue Total RNA 100 ng 54,787 Breast Tissue Total RNA 10 ng5,486 Lung Tissue Total RNA 100 ng 2,319 Negative Control for Total RNA0 1,620 Prostate Tissue Poly-A RNA 10 ng 2,510,601 Peripheral BloodPoly-A RNA 10 ng 1,811 Breast Tissue Poly-A RNA 10 ng 120,264 KidneyTissue Poly-A RNA 10 ng 147,284 Liver Tissue Poly-A RNA 10 ng 1,900Negative Control for Poly-A RNA 0 1,586 PSA In vitro Transcripts 10,000copies 1,309,171 PSA In vitro Transcripts 1,000 copies 218,205 PSA Invitro Transcripts 100 copies 21,421 PSA In vitro Transcripts 10 copies3,771

In separate experiments, poly-A RNA isolated from human prostate tissue,peripheral blood, breast tissue, kidney tissue, liver tissue, smallintestine and lymph node were compared in similar amplification anddetection assays, using the same combination of primers and probe asabove. The mRNA for prostate tissue was assayed at 5 fg or 0.5 fg perreaction, and the mRNA from the other biological sources was assayed at5 ng and 0.5 ng per reaction. Under these conditions, PSA-specifictarget sequences were detected in the prostate tissue, breast tissue,kidney tissue, small intestine and lymph node, but no signal above thenegative control (i.e., reactions without added RNA) was detected inliver tissue or blood. The relative signal (mean RLU of duplicateassays) detected indicated that the PSA target nucleic acid present inbreast tissue, kidney tissue, small intestine and lymph node was about10⁵-fold to 10⁶-fold less than present in prostate tissue.

These results show that the PSA target detection system can detect evenrelatively small amounts of target in tissues other than prostatetissue. Because some tested tissues (liver and peripheral blood) werenegative for PSA-specific target in these assays indicates that the PSAgene is not merely expressed at a lower level in all human tissuesbesides prostate. Therefore, the PSA target may be a general marker forcancerous or pre-cancerous conditions in selected other human tissues.An increase in detectable PSA-specific target (e.g., in peripheralblood) may then be an indication of metastasized prostate cancer orother types of cancer.

EXAMPLE 9 Amplification and Detection of PSA, PSMA and hK2 Targets inVarious Biological Samples

In addition to PSA, the other prostate-associated cancer marker targetsPSMA and hK2 are also useful for detecting the presence of these targetmRNA in non-prostate tissue. Here, amplification and detection assayswere performed on total and poly(A) RNA isolated from human tissues todetermine the relative levels of PSA, PSMA and hK2 specific target inthose tissues. All tissues were obtained from normal donors (i.e.,non-cancerous tissue) and the assays were performed in triplicate foreach sample, substantially as described in Example 8. For each testedsample, the known amount of RNA or poly(A) RNA was as indicated in Table10, where 5 ng of poly(A) RNA is equivalent to about 100 ng of totalRNA. For PSA detection, transcription-mediated amplification wasperformed using a non-T7(+) primer having SEQ ID NO:18, a T7(−)promoter-primer having SEQ ID NO:30 and an AE-labeled probe having SEQID NO:1. For PSMA detection, transcription-mediated amplification wasperformed using a non-T7(+) primer having SEQ ID NO:48, a T7(−)promoter-primer having SEQ ID NO:49 and an AE-labeled probe having SEQID NO:50. For hK2 detection, transcription-mediated amplification wasperformed using a non-T7(+) primer having SEQ ID NO:46, a T7(−)promoter-primer having SEQ ID NO:47 and an AE-labeled probe having SEQID NO:1. Detection of RLU was substantially as described above. The meanRLU results of the assays performed on triplicate samples for eachresult are shown in Table 10 (ND means “not done”).

TABLE 10 Detection of Prostate-Associated Genetic Marker Targets inBiological Samples Tissue/RNA type RNA Amount PSA hK2 PSMA Prostate 1 ng2.58 × 10⁶ 1.37 × 10⁶ 1.19 × 10⁶ Total RNA 100 pg 2.54 × 10⁶ 3.81 × 10⁵7.71 × 10⁴ 10 pg 2.40 × 10⁶ 1.54 × 10⁴ 8.97 × 10³ 1 pg 2.18 × 10⁶ 2.15 ×10³ 2.08 × 10³ 100 fg 1.87 × 10⁶ 1.59 × 10³ 1.76 × 10³ 10 fg 6.22 × 10⁵ND ND 1 fg 1.68 × 10³ ND ND Blood 100 ng 2.12 × 10³ 1.39 × 10³ 1.57 ×10³ Total RNA 10 ng 1.71 × 10³ 1.27 × 10³ 1.51 × 10³ Breast 100 ng 2.78× 10⁶ 1.18 × 10⁴ 2.04 × 10⁶ Total RNA 10 ng 1.56 × 10⁶ 3.14 × 10³ 1.13 ×10⁶ 1 ng 1.17 × 10⁶ ND 2.57 × 10⁴ 0.1 ng 1.84 × 10³ ND 4.46 × 10³ Lung100 ng 1.94 × 10⁵ 1.29 × 10³ 1.85 × 10⁶ Total RNA 10 ng 4.27 × 10³ 1.37× 10³ 3.16 × 10⁶ 1 ng 1.76 × 10³ ND 7.20 × 10³ 0.1 ng ND ND 2.61 × 10³Lymph Node 5 ng 2.73 × 10⁶ 1.59 × 10⁵ 2.14 × 10⁶ Poly(A) RNA 500 pg 2.52× 10⁶ 1.68 × 10⁴ 1.92 × 10⁵ 50 pg 7.89 × 10⁵ 2.05 × 10³ 6.84 × 10³ 5 pg1.66 × 10³ 1.33 × 10³ 3.22 × 10³ Kidney 5 ng 2.60 × 10⁶ 1.24 × 10³ 2.54× 10⁶ Poly(A) RNA 500 pg 1.04 × 10⁶ 1.29 × 10³ 8.01 × 10⁵ 50 pg 1.29 ×10⁵ ND 7.37 × 10⁴ 5 pg 1.66 × 10³ ND 4.31 × 10³ Small Intestine 5 ng2.70 × 10⁶ 1.42 × 10³ 1.34 × 10⁶ Poly(A) RNA 500 pg 6.40 × 10⁵ 2.28 ×10³ 1.73 × 10⁶ 50 pg 4.89 × 10⁴ ND 1.18 × 10⁵ 5 pg 3.64 × 10³ ND 8.32 ×10³

The results shown in Table 10 indicate that the amplification anddetection methods used here detected PSA-specific RNA in a prostatesample containing at least 10 fg of total RNA, in a breast samplecontaining at least 1 ng of total RNA, in a lung sample containing atleast 100 ng of total RNA, and in samples containing at least 50 pgpoly(A) RNA isolated from lymph node, kidney and small intestine. ForhK2, the assays detected this genetic marker RNA in prostate total RNA(at least 10 pg/sample), breast total RNA (at least 100 ng/sample), andlymph node poly(A) RNA (at least 500 pg/sample). For PSMA, the assaysdetected this genetic marker RNA in prostate total RNA (at least 10pg/sample), breast total RNA (at least 1 ng/sample), lung (at least 1ng/sample) and in poly(A) RNA isolated from lymph node (at least 50pg/sample), kidney (at least 50 pg/sample) and small intestine (at least5 pg/sample). None of the primer and probe combinations detected thegenetic marker (PSA, hK2, PSMA) in RNA isolated from normal blood, andgenerally hK2 was detected in lower amounts in the other tissuesrelative to PSA or PSMA. Table 10 results also show that assays arequantitative, detecting RLU that are relatively proportional to theamount of target-specific RNA in the sample, up to the saturation pointof detection (about 2.5×10⁶ RLU in these experiments). Thus standardtitration experiments could be used to quantitatively determine therelative amounts of target RNA in a sample.

EXAMPLE 10 Amplification and Detection of PSA, PSMA and hK2 Targets inClinical Samples

In this example, clinical samples (peripheral blood drawn from patientshaving benign prostate hyperplasia (BPH) or prostate carcinoma (CaP), orblood drawn from normal controls) were tested in a blinded study wherethe person performing the assays did not know the source of the testedsample. Total RNA was isolated from the clinical samples using standardguanidium isothiocyanate extraction methods (Sambrook, J. et al., 1989,Molecular Cloning, A Laboratory Manual, 2^(nd) ed. (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.), pp. 7.37-7.57). Foramplification using TMA and detection substantially as described earlierherein, each sample was assayed in duplicate using 500 ng for detectionof PSA RNA, 500 ng for detection of hK2 RNA and 100 ng for detection ofPSMA. Positive samples were those that produced detectable signal abovea cutoff point of greater than 5,000 RLU for PSA detection, greater than10,000 RLU for hK2 detection, and greater than 1,500 RLU for PSMAdetection; negative samples were those that produced signal below thecutoff points for each target. The primers and probes were the same asused in the assays described in Example 9 (for PSA, SEQ ID Nos 18, 30and 1; for PSMA, SEQ ID NOs 48, 49 and 50; and for hK2, SEQ ID NOs 46,47 and 1). The results of these assays are shown in Table 11, with thespecimens grouped according to the disease state of the person from whomthe sample was taken.

TABLE 11 Detection of Prostate-associated Genetic Markers in ClinicalSamples Specimen No. Disease State PSA Target hK2 Target PSMA Target 1CaP positive negative positive 2 CaP positive negative negative 3 CaPpositive negative negative 4 CaP positive negative negative 5 CaPpositive positive negative 6 CaP positive positive negative 7 CaPpositive negative negative 8 CaP positive positive positive 9 CaPpositive negative negative 10 CaP negative negative positive 11 CaPpositive positive negative 12 CaP positive positive negative 28 CaPnegative positive negative 29 CaP negative positive negative 30 CaPnegative negative negative 13 Normal Control negative negative negative14 Normal Control negative negative negative 15 Normal Control negativenegative negative 16 Normal Control negative negative positive 17 NormalControl negative positive negative 18 BPH negative negative negative 19BPH negative negative negative 20 BPH positive positive negative 21 BPHnegative negative negative 21 BPH negative negative negative 23 BPHnegative negative positive 24 BPH negative positive negative 25 BPHnegative negative positive 26 BPH negative positive negative 27 BPHnegative negative negative

The results of Table 11 show a generally positive correlation betweenthe clinical status of the sample donor and the detection of PSA. Elevenof fourteen prostate cancer patients were positive, only one of ten BPHpatients was positive and none of the normal controls was positive inthe assay. Detection of hK2 also showed a positive correlation toprostate carcinoma status (7 of 14 were positive), although one normalcontrol was positive and three BPH patients of ten were positive.Detection of PSMA showed no strong correlation with the clinical statusof the donor, but for two of the three prostate cancer patients thattested positive for PSMA, they were also positive for PSA. One prostatecancer patient (specimen 10) was positive only for PSMA and two prostatecancer patients were positive only for hK2. These results show that theassays specifically detect the individual cancer genetic markers in RNAisolated from clinical samples and that each marker individually, or incombination with one or two other prostate-associated genetic cancermarker, is useful for detecting a molecular marker that correlates withprostate cancer. For patients that show symptoms of prostate cancer buttest negative with one genetic marker (e.g., PSA), another marker (e.g.,hK2) may result in a positive signal, thus decreasing the potential of afalse negative diagnosis that would result from reliance on results ofonly one marker. For prostate cancer patients that test positive for oneprostate-associated genetic marker (e.g., PSA), a positive result obtainfor another genetic marker (e.g., PSMA or hK2) may be used to furtheradd support to the positive diagnosis or to provide an indication of theextent of tumor growth. In all cases, because the positive results wereobtained with non-prostate samples, the results may support thediagnosis that metastasis has occurred.

The examples presented herein are meant to more fully describe preferredembodiments of the present invention which is defined by the claims thatfollow. All embodiments that are legal equivalents of the invention areanticipated to be within the scope of the claims.

1. A combination of oligonucleotides used in a detection assay specificfor a prostate specific antigen (PSA) target nucleic acid sequence,comprising: a first oligonucleotide comprising a target-binding sequenceof SEQ ID NO:40 (ACCCAGCAAGATCACGCTTTTG) or its equivalent RNA, or afully complementary base sequence of the target-binding sequence of SEQID NO:40 or its equivalent RNA, that serves as a first amplificationprimer that hybridizes specifically to a first PSA-specific sequencecontained in exon 3 of a PSA expressed gene sequence; a secondoligonucleotide that serves as a second amplification primer thathybridizes specifically to a different, non-overlapping secondPSA-specific sequence contained in exon 2 of a PSA expressed genesequence; and a third oligonucleotide comprising SEQ ID NO:8(ACAGCTGCCCACTGCATCAGG) or its equivalent RNA, or a fully complementarybase sequence of SEQ ID NO:8 or its equivalent RNA, that serves as adetection probe that hybridizes specifically to a third PSA-specificsequence contained in exon 2 of a PSA expressed gene sequence.
 2. Thecombination of oligonucleotides in claim 1, wherein the secondoligonucleotide comprises SEQ ID NO:21 (TCTCGTGGCAGGGCAGTCTGC) or itsequivalent RNA, or a fully complementary base sequence of SEQ ID NO:21or its equivalent RNA.
 3. The combination of oligonucleotides in claim1, wherein the second oligonucleotide comprises SEQ ID NO:26(GCAGTCTGCGGCGGTGTTCTG) or its equivalent RNA, or a fully complementarybase sequence of SEQ ID NO:26 or its equivalent RNA.
 4. A combination ofoligonucleotides used in a detection assay specific for a prostatespecific antigen (PSA) target nucleic acid sequence, comprising: a firstoligonucleotide that includes a sequence consisting of a target-bindingsequence of SEQ ID NO:40 (ACCCAGCAAGATCACGCTTTTG) or its equivalent RNA,or a fully complementary base sequence of the target-binding sequence ofSEQ ID NO:40 or its equivalent RNA, that serves as a first amplificationprimer that hybridizes specifically to a first PSA-specific sequencecontained in exon 3 of a PSA expressed gene sequence; a secondoligonucleotide that serves as a second amplification primer thathybridizes specifically to a different, non-overlapping secondPSA-specific sequence contained in exon 2 of a PSA expressed genesequence; and a third oligonucleotide consisting of SEQ ID NO:8(ACAGCTGCCCACTGCATCAGG) or its equivalent RNA, or a fully complementarybase sequence of SEQ ID NO:8 or its equivalent RNA, that serves as adetection probe that hybridizes specifically to a third PSA-specificsequence contained in exon 2 of a PSA expressed gene sequence.
 5. Thecombination of oligonucleotides in claim 4, wherein the secondoligonucleotide consists of SEQ ID NO:21 (TCTCGTGGCAGGGCAGTCTGC) or itsequivalent RNA, or a fully complementary base sequence of SEQ ID NO:21or its equivalent RNA.
 6. The combination of oligonucleotides in claim4, wherein the second oligonucleotide consists of SEQ ID NO:26(GCAGTCTGCGGCGGTGTTCTG) or its equivalent RNA, or a fully complementarybase sequence of SEQ ID NO:26 or its equivalent RNA.
 7. A method ofdetecting a prostate-associated target nucleic acid in a biologicalsample containing nucleic acid, comprising the steps of: providing anucleic acid sample containing a target nucleic acid that includes atleast a portion of an expressed gene sequence encoding prostate-specificantigen (PSA); hybridizing to the target nucleic acid a firstoligonucleotide comprising a target-binding sequence of SEQ ID NO:40(ACCCAGCAAGATCACGCTTTTG) or its equivalent RNA, or a fully complementarybase sequence of the target-binding sequence of SEQ ID NO:40 or itsequivalent RNA, and an optional promoter sequence adjacent to thetarget-binding sequence, that serves as a first amplification primer;hybridizing to the target nucleic acid or a fully complementary strandof the target nucleic acid a second oligonucleotide that serves as asecond amplification primer that hybridizes specifically to a different,non-overlapping second PSA-specific sequence contained in exon 2 of aPSA expressed gene sequence; producing a plurality of amplificationproducts of the target nucleic acid by using the first and secondamplification primers and at least one polymerase activity; providing aprobe oligonucleotide comprising SEQ ID NO:8 (ACAGCTGCCCACTGCATCAGG) orits equivalent RNA, or a fully complementary base sequence of SEQ IDNO:8 or its equivalent RNA, that hybridizes specifically to at least oneamplification product of the target nucleic acid; and detecting a signalresulting from the probe hybridized to the amplification product.
 8. Themethod of claim 7, wherein the second oligonucleotide comprises SEQ IDNO:21 (TCTCGTGGCAGGGCAGTCTGC) or its equivalent RNA, or a fullycomplementary base sequence of SEQ ID NO:21 or its equivalent RNA. 9.The method claim 7, wherein the second oligonucleotide comprises SEQ IDNO:26 (GCAGTCTGCGGCGGTGTTCTG) or its equivalent RNA, or a fullycomplementary base sequence of SEQ ID NO:26 or its equivalent RNA.
 10. Amethod of detecting a prostate-associated target nucleic acid in abiological sample containing nucleic acid, comprising the steps of:providing a nucleic acid sample containing a target nucleic acid thatincludes at least a portion of an expressed gene sequence encodingprostate-specific antigen (PSA); hybridizing to the target nucleic acida first oligonucleotide that includes a sequence consisting of atarget-binding sequence of SEQ ID NO:40 (ACCCAGCAAGATCACGCTTTTG) or itsequivalent RNA, or a fully complementary base sequence of thetarget-binding sequence of SEQ ID NO:40 or its equivalent RNA, and anoptional promoter sequence adjacent to the target-binding sequence, thatserves as a first amplification primer; hybridizing to the targetnucleic acid or a fully complementary strand of the target nucleic acida second oligonucleotide that serves as a second amplification primerthat hybridizes specifically to a different, non-overlapping secondPSA-specific sequence contained in exon 2 of a PSA expressed genesequence; producing a plurality of amplification products of the targetnucleic acid by using the first and second amplification primers and atleast one polymerase activity; providing a probe oligonucleotideconsisting of SEQ ID NO:8 (ACAGCTGCCCACTGCATCAGG) or its equivalent RNA,or a fully complementary base sequence of SEQ ID NO:8 or its equivalentRNA, that hybridizes specifically to at least one amplification productof the target nucleic acid; and detecting a signal resulting from theprobe hybridized to the amplification product.
 11. The method of claim10, wherein the second oligonucleotide consists of SEQ ID NO:21(TCTCGTGGCAGGGCAGTCTGC) or its equivalent RNA, or a fully complementarybase sequence of SEQ ID NO:21 or its equivalent RNA.
 12. The method ofclaim 10 wherein the second oligonucleotide consists of SEQ ID NO:26(GCAGTCTGCGGCGGTGTTCTG) or its equivalent RNA, or a fully complementarybase sequence of SEQ ID NO:26 or its equivalent RNA.